In-vivo and in-vitro study of mechanism ofaction of 4 hydroxyisoleucine as an amino acidderived from fenugreek seed with anti-diabetic
and properties
A thesis submitted for the Degree of Doctor of PhilosophyBy Hamidreza Khalatbari Limaki
London Metropolitan University
Faculty of Life Science and Computing
April 2014
Author's declaration
I attest that the work presented in this thesis entitled “ In-vivo and in-vitro study of mechanism of action of 4 hydroxyisoleucine as an aminoacid derived from fenugreek seed with anti-diabetic properties” has notbeen submitted in support of any qualification in this or othereducational institutions in the UK or elsewhere.
Hamidreza Khalatbari Limaki
Date : April 2014
Table of contents
Acknowledgement ............................................................................. I
Abstract .............................................................................................. III
Chapter I (Introduction) ................................................................... 1
1.1 Type 1 DM …......................…....................................................................... 51.2 Type 2 DM …................................................................................................. 61.3 Diabetes complications ….............................................................................. 91.4 Diagnosis criteria …....................................................................................... 111.5 Insulin …......................................................................................................... 1.5.1 Structure …................................................................................................. 1.5.2 Secretion …................................................................................................ 1.5.3 Mechanism of Action of Insulin …............................................................
13131416
1.6 Glucose transporters …................................................................................... 211.7 Insulin resistance …........................................................................................ 251.8 Diabetes management and available therapeutics …...................................... 331.9 Fenugreek (4-Hydroxyisoleucine) …............................................................. 361.10 Isoleucine …................................................................................................... 401.11 Background (4-Hydroxyisoleucine) ............................................................... 421.12 Background (Isoleucine) …............................................................................ 491.13 Summary …..............................….................................................................. 511.14 Questions and study design notions …........................................................... 54
Chapter II (Materials and Methods) ................................................ 57
Part I (Animal Trial) ….................................................................... 58
2.1 Animals …....................................................................................................... 592.2 Preliminary study …........................................................................................ 592.3 Main Experiment …......................................................................................... 602.4 Insulin Assay …............................................................................................... 612.5 Serum lipid profile, glucose and uric acid …................................................... 622.6 Statistical analysis …........................................................................................ 63
Part II (Laboratory In-vitro studies) ….......................................... 64
2.7 Cell line and cell culture ….............................................................................. 652.8 Glucose measurement ….................................................................................. 662.9 ATP assay …..................................................................................................... 662.10 Insulin measurement ….................................................................................... 672.11 Compounds ….................................................................................................. 682.12 Cell metabolism and mitochondrial respiration analysis …............................. 712.13 Optimisation of cell number for Seahorse XF-24 …........................................ 722.14 Results of cell number optimization …............................................................ 74
Chapter III (Animal Trial) ................................................................. 76
3.1 Type 1 diabetes animal model …..................................................................... 773.2 Animal study results and discussion …............................................................ 813.3 Animal study findings at a glance .................................................................... 92
Chapter IV (In-vitro study, BRIN-BD11 cell) ..........................................
4.1 Introduction ….................................................................................................. 4.1.1 BRIN-BD 11 cell as a model for In-vitro study.............................................
93
94
95
4.2 The effect of 4-hydroxyisoleucine and isoleucine on glucose consumption in BRIN-BD11 cells ............................................................................................. 98
4.3 The effect of 4-hydroxyisoleucine and isoleucine on insulin secretion from BRIN-BD11 cells …................................................................................. 100
4.4 The effect of 4-hydroxyisoleucine and isoleucine on ATP content of BRIN-BD11 cells …......................................................................................... 102
4.5 Cycloheximide (CHX) and protein synthesis role in 4-hydroxyisoleucine and isoleucine mechanism of actions…............................................................ 105
4.6 Wortmannin and PI3Kinase role in 4-hydroxyisoleucine and isoleucine activities in BRIN-BD11cells …...................................................................... 107
4.7 Wortmannin and Cycloheximide (CHX) increase glucose consumption in BRIN-BD11 cells …......................................................................................... 109
4.8 The mTOR role in 4-hydroxyisoleucine and isoleucine mechanism of actions …....................................................................................................... 111
4.9 Role of GLUT1 in 4-hydroxyisoleucine and isoleucine mechanism of actions in BRIN-BD11 cells ............................................................................. 115
4.10 The role of calcium on the effects of 4-hydroxyisoleucine and isoleucine on glucose consumption rate and ATP content in BRINBD11cells …............. 118
4.11 The effect of the UK-5099 as a mitochondrial pyruvate carrier (MPC) inhibitor on 4-hydroxyisoleucine and isoleucine glucose utilisation in BRIN-BD11cells .............................................................................................. 130
4.12 Analysis of the effects of 4-hydroxyisoleucine and isoleucine on mitochondrial respiration with Seahorse XF-24 autoanalyser …..................... 134
Chapter V (Conclusions) ......................................................................
5.1 Future recommended studies ….......................................................................
139
153
References ….......................................................................................... 156
Appendix I .............................................................................................• Animal study publication. (Non-insulin dependent anti-diabetic activity of
(2S, 3R, 4S) 4-hydroxyisoleucine of fenugreek (Trigonella foenum graecum) in streptozotocin-induced type I diabetic rats)
192
Appendix II …........................................................................................• 4-Hydroxyisoleucine certificate of analysis
197
Appendix II …........................................................................................• Glucometer accuracy measurements table
202
Acknowledgements
I am very grateful to Professor Christopher Branford-White and Dr
Kenneth White for giving me the opportunity to work and carry out my
research in Institute of Health and Policy (IHRP) at London
Metropolitan University. Their scientific knowledge, enthusiasm,
passion, countless support and precious advise were a great help in my
research and scientific journey.
I would like to express my gratitude to Dr Mohammad Reza Haeri who
brought the idea for my research and provided me the facility to run the
animal study in his laboratory at the University of Qom, Medical
School in Iran. He helped me to develop my research skills and without
his support this work would not have been achievable.
I thank my colleague, Ravi Velga for his great help and excellent
cooperation to run the experiments and overcome the problem with his
great scientific knowledge.
Many thanks to the members of technical support staff for ensuring the
smooth running of all activities in the science centre.
I show all my gratitude to my parents for their support and
encouragement. They sponsored all my research expenses in this
presented work and persistently supported me financially and
emotionally in all the conditions and situations.
I am deeply grateful to my wife, Leila Razavi for her moral and
emotional supports. Her encouragement and understanding have given
I
me confidence and motivation to continue my work and overcome the
problems and obstacles. I believe that without her continuous help I
could not have completed this work. Thank you with all my heart for
always taking care of me and love me.
II
Abstract
Diabetes is a progressive multi-factorial metabolic syndrome with
serious short and long term complications affecting many of organs
with high increasing prevalence in the world. Using herbs and their
derivatives for treating diabetes has a long history in many traditional
cultures across the world. Molecules and compounds were isolated
from herbs are the basis of many therapeutics which we are using in
medicine for treating a variety of health conditions. The seeds of
fenugreek, Trigonella foenum graecum, commonly used as a spice in
Middle Eastern countries and widely used in South Asia and Europe,
are known to have anti-diabetic properties. In 1979, Hardman
identified an unusual amino acid (2S, 3R, 4S) 4-hydroxyisoleucine
(4HO-Ile) in a fenugreek seed extract as an active compound in
fenugreek seed. It was so far found only in fenugreek seed, which is
responsible for its anti-diabetic properties. Studies on 4-
hydroxyisoleucine effects on type 2 diabetes and insulin resistant
animal models revealed that it has anti-diabetic properties of enhancing
insulin secretion under hyperglycaemic conditions, and increasing
insulin sensitivity. Unfortunately, the available published researches for
4-hydroxyisoleucine are limited and its mechanism of actions is not
clear. Here we describe for the first time the anti-diabetic activity of 4-
hydroxyisoleucine in a model of type 1 diabetes as all the previous
works focused on 4-hydroxyisoleucine activity in type 2 diabetes and
insulin resistant condition. Treatment of streptozotocin-treated type 1
diabetes rats, where levels of insulin are much reduced, by 65%,
compared to normal animals, with daily doses of 4-hydroxyisoleucine
at 50 mg/kg/day for four weeks could reduce plasma glucose in the
diabetic group. Moreover the high levels of lipids (cholesterol, HDL,
III
LDL and triglyceride) and uric acid in the diabetic rats, could be
restored to levels found in non-diabetic controls by the treatment with
4-hydroxyisoleucine. These results demonstrate that 4-
hydroxyisoleucine has significant anti-diabetic activities in type 1
diabetes as well as previously studied type 2 diabetes and insulin
resistance model that are independent of insulin. The findings suggest
the potential of 4HO-Ile as an adjunct to diabetes treatment and for type
1 as well as type 2 diabetes.
To investigate the insulin-independent effects of 4-hydroxyisoleucine
further, the cell based experiments were designed to assess the effect
of 4-hydroxyisoleucine on cellular glucose uptake and ATP content
after one day incubation. Isoleucine was added to the experiment as a
positive control because firstly it has some level of anti-diabetic
properties according to previously published studies and secondly it
has similar molecular backbone as 4-hydroxyisoleucine. BRIN-BD
11, a functional and glucose responsive pancreatic beta cell, was
selected as a cell model which is not insulin-responsive and dependent
on the insulin signalling pathway for glucose uptake. Use of the model
provides the opportunity to study the mechanisms of action of both 4-
hydroxyisoleucine and isoleucine independently. We adopted a unique
approach using inhibitors to target suggested pathway and molecules
within the cell which may be involved in both 4-hydroxyisoleucine and
isoleucine mechanism of actions. The results revealed that 4-
hydroxyisoleucine and isoleucine could increase glucose uptake in
BRIN-BD 11 cells, but as previously suggested, 4-hydroxyisoleucine
activity is in direct correlation with glucose concentration. 4-
hydroxyisoleucine has higher activity in higher concentrations of
glucose. 4-hydroxyisoleucine increased the glucose uptake much
greater than isoleucine at 11mM and 22mM concentration of glucose in
IV
cell culture medium. Endpoint measurements of ATP content of the
BRIN-BD11 cells did not show any significant changes between 4-
hydroxyisoleucine and isoleucine groups and control as well as an
insulin level measurement in culture medium after 24 hours. The
results showed that there are substantial differences between
isoleucine and 4-hydroxyisoleucine mechanisms of action unlike
their similar glucose uptake stimulatory effect which is greater in 4-
hydroxyisoleucine. 4-hydroxyisoleucine activity strongly dependent
on new protein synthesis and GLUT 1 activity. GLUT 1 is widely
available in most of the cells and it controls the basal glucose uptake
independent of insulin. The connection between GLUT 1 and 4-
hydroxyisoleucine effect in cellular level, supports the idea that 4-
hydroxyisoleucine utilises the glucose basal consumption and uptake
of cells. Mitochondrial calcium channel signalling inhibition affects
4-hydroxyisoleucine and isoleucine functionality as well as
inhibition of mitochondria pyruvate carrier. Real time monitoring of
cell metabolism by Seahorse XF-24 autoanalyser after 24 hours
incubation with 4-hydroxyisoleucine and isoleucine revealed that
both 4-hydroxyisoleucine and isoleucine activities are strongly
dependent on mitochondrial respiration but 4-hydroxyisoleucine
significantly up-regulates glycolysis which is not affected by
isoleucine. The connection between mitochondria calcium signalling
and contradictory behaviour of 4-hydroxyisoleucine and isoleucine
support the very important role of mitochondria in their mechanisms
of action.
V
Chapter I
Introduction
1
Diabetes mellitus refers to a group of metabolic diseases defined by
hyperglycaemia (high blood sugar) and other classical symptoms such as
polyuria (frequent urination), polydipsia (increased thirst) and
polyphagia (increased hunger). Diabetes is classified into four main
types based on the pathogenic process which creates hyperglycaemia
including Type 1, Type 2, Other Specific Types known as MODYs
(Maturity onset diabetes of the young) and Gestational Diabetes (Fig
1.1).
Type 1 diabetes is result of insulin deficiency which pancreatic cells can
not produce and secrete insulin. Type 2 diabetes is a group of progressive
disorders characterized by insulin resistance, impaired insulin secretion
and increased gluconeogenesis (production of glucose). Diabetes can
happen with other types of pathogenesis, such as exocrine disease of the
pancreas, genetic defects in insulin secretion, mitochondrial
abnormalities and mutation in the insulin receptor, which are categorized
as Other Specific Type. Gestational Diabetes refers to glucose
intolerance during pregnancy due to metabolic changes of pregnancy and
increased insulin demands.
Diabetes is one of the main public health concerns as a major cause of
mortality in most countries with a high prevalence rate, which has
increased dramatically in the last two decades (Fig 1.2).
2
Fig 1.1. Spectrum of glucose homeostasis and diabetes mellitus (DM). The
spectrum from normal glucose tolerance to diabetes in type 1, type 2, other specific
types of diabetes, and gestational diabetes is shown from left to right. In most types
of diabetes, the individual traverses from normal glucose tolerance to impaired
glucose tolerance to overt diabetes (these should be viewed not as abrupt categories
but as a spectrum). Arrows indicate that changes in glucose tolerance may be
bidirectional in some types of diabetes. For example, individuals with type 2 diabetes
may return to the impaired glucose tolerance category with weight loss; in gestational
diabetes, diabetes may revert to impaired glucose tolerance or even normal glucose
tolerance after delivery. The fasting plasma glucose (FPG), the 2-h plasma glucose
(PG) after a glucose challenge, and the A1C (Glycated hemoglobin or glycosylated
hemoglobin or HbA1c) for the different categories of glucose tolerance are shown in
the lower part of the figure. These values do not apply to the diagnosis of gestational
diabetes. The World Health Organization uses an FPG of 110–125 mg/dL for the
prediabetes category. Some types of diabetes may or may not require insulin for
survival. *Some use the term "increased risk for diabetes" (ADA, American Diabetes
Associassion) or "intermediate hyperglycaemia" (WHO) rather than "prediabetes."
(Adapted from the American Diabetes Association, 2007.)
3
Fig 1.2. Worldwide prevalence of diabetes mellitus. Comparative prevalence (% of
total population) and estimates numbers of diabetes patients (20–79 years), 2010.
(Used with permission from IDF Diabetes Atlas, the International Diabetes
Federation, 2000 Adopted from Harrison's Principles of internal medicine, 18th
Edition)
Prevalence of Type 2 diabetes is increasing more rapidly due to
increasing obesity, reducing physical activity and industrialisation. The
increasing rate of diabetes will place a burden of large costs to societies
which puts this disease at the centre of attention for all public health
policy makers, researchers and medical groups. The number of people
with diabetes in the UK has increased from 1.4 million in 1996 to 2.9
million and most of the cases is Type 2 diabetes (www.diabetes.org.uk).
The figures announced by the WHO and the UK health authorities show
that diabetes, particularly Type 2 diabetes, is one of the biggest health
challenges in the world today. The seriousness and importance of this
issue make diabetes research more in demand in order to find new
solutions to reduce the rate of diabetes prevalence and new treatments to
control diabetes and its complications.
4
1.1 Type 1 Diabetes
Type 1 diabetes is characterised by total insulin deficiency as a result of
destruction of pancreatic beta cells by interactions of genetic,
environmental and immunological (autoimmune pancreatic cell
destruction) factors (Harrison's Principles of internal medicine, 18th
Edition). The main pathophysiology of type 1 diabetes is the activation of
an innate immune system response towards pancreatic beta cells,
involving expansion of auto-reactive immune system cells such as CD4 ,
CD8 and T-Helper cells and producing auto-antibodies against beta cells
(Bluestone et al. 2010). Multiple genes are involved in susceptibility to
type 1 diabetes. The major susceptibility gene for type 1 diabetes is
located in the HLA region on chromosome 6 which account for 40–50%
of the genetic risk of developing type 1 diabetes (Bluestone et al. 2010).
In addition to MHC class II associations, genome association studies
have identified at least 20 different genetic loci that contribute
susceptibility to type 1 diabetes, such as polymorphisms in the promoter
region of the insulin gene, the CTLA-4 gene, interleukin-2 receptor,
CTLA4, and PTPN22, etc. (Grant et al. 2009).
Type 1 diabetes is fatal and should be treated by injecting appropriate
amounts of insulin to control high blood sugar levels. Type 1 diabetes
can be distinguished from type 2 diabetes by measuring endogenous
insulin production using the C-Peptide assay. Diabetic ketoacidosis
(DKA) is the most important acute complication of untreated or poorly
controlled type 1 diabetes and which needs special medical attention.
DKA develops when the ratio of insulin to glucagon becomes decreased
in a severe insulin deficiency condition, leading to ketone body
formation in the liver and create acidosis, electrolyte imbalance and
5
metabolic complications (Harrison's Principles of internal medicine, 18th
Edition).
1.2 Type 2 Diabetes
Type 2 diabetes is the most common form of diabetes and has genetic
predisposing factors. It is characterized by insulin resistance, impaired
insulin secretion, obesity and abnormal fat metabolism (Harrison's
Principles of internal medicine, 18th Edition). Insulin resistance is the
main underlying pathogenesis of type 2 diabetes, and in turn leads to
impaired insulin secretion and other metabolic disturbances. A glucose
tolerance test is normal or near normal in the early stage of disease
because pancreatic beta cells compensate insulin resistance by increasing
insulin secretion (Harrison's Principles of internal medicine, 18th
Edition).
It is expected that the level of serum insulin increases (hyperinsulinemia)
in the early stages of type 2 diabetes as a response to impaired insulin
action (Stefano Del Prato et al. 2002). Continuous stimulation of beta
pancreatic cells to keep the compensatory output of insulin high leads to
impaired insulin secretion as a result of exhaustion of the cells and
failure to produce insulin (Fig 1.3).
Type 2 diabetes risk increases with a positive familial history due to the
strong genetic component with concordance between 70% and 90% in
identical twins (Harrison's Principles of internal medicine, 18th Edition).
The risk of type 2 diabetes can increase up to 40% if both parents are
diabetic. Apart from genetic factors, environmental factors such as food
intake, and exercise play an important role in developing diabetes.
Studies show that there is a connection between consumption of sugar
6
sweetened drinks and risk of type 2 diabetes (Malik et al. 2010).
Lifestyle and diet modification, are considered as a very effective
preventive approach, especially for those people with higher risk,
including people with obesity and a positive familial history for diabetes.
NICE guide line recommends bariatric surgery for people with type 2
diabetes with BMI higher than 35 kg/m2 as effect intervention to control
obesity and diabetes. A recent study showed that bariatric surgery can
significantly improve type 2diabetes and other metabolic risk factors in
severely obese patients (Brethauer et al. 2013).
Fig 1.3. Metabolic changes during the development of type 2 diabetes mellitus (DM).
Insulin secretion and insulin sensitivity are related, and as an individual becomes
more insulin resistant (by moving from point A to point B), insulin secretion
increases. A failure to compensate by increasing the insulin secretion results initially
in impaired glucose tolerance (IGT; point C) and ultimately in type 2 diabetes (point
D). (Adapted from SE Kahn: J Clin Endocrinol Metab 86:4047, 2001; RN Bergman,
M Ader: Trends Endocrinol Metab 11:351, 2000) M Value is a score developed by
McCauley et al in 2001 for measuring the Insulin Sensitivity Index (ISI) by the
euglycemic-hyperinsulinemic glucose clamp technique. The ISI is calculated for fat-
free body mass by dividing the whole body glucose disposal rate (M – mg/min or
µmol/min per Kg) by the average plasma insulin concentration over the final 60
minutes of the 120 minute test (McAuley et al. 2001).
7
Many genes are involved in diabetes with subtle variation known as
single nucleotide polymorphism (SNPs) which increase the risk of
developing diabetes. Early whole genome linkage studies identified
calpain 10 (CAPN10) and hepatocyte nuclear factor 4 alpha (HNF4A) as
directly linked to diabetes (Dean et al. 2004). More than 36 genes have
been identified in relation to the risk of type 2 diabetes (Herder et al.
2011). Genome-wide association studies (GWAS) have identified novel
pathways linked to fundamental biology, which confirms previous
epidemiological observations, showed the role of β-cell dysfunction in
type 2 diabetes, explained about 10% of disease heritability, and
determined possible targets for pharmacotherapy (Billings et al. 2010).
GWAS has discovered the variant rs13266634, which encodes a R→W
change at position 325 in the beta cell zinc transporter ZnT-8 which
encoded by SLC30A8 gene with strong associations with type 2 diabetes
(Scott et al. 2007). The GWAS method provided potential genetic links
between lipid dysregulation and glycemia (FADS1, GCKR, HNF1A),
circadian rhythmicity and metabolic derangements (MTNR1B, CRY2),
and low birth weight with subsequent type 2 diabetes risk (ADCY5)
(Billings et al. 2010). The genomic study revealed that the risk of
developing diabetes, especially type 2 diabetes is a combination of
genetic risk for pancreatic beta cell dysfunction superimposed on genetic
and environmental factors (e.g. obesity, Western diet, sedentary lifestyle)
that promote insulin resistance.
8
1.3 Diabetes Complications
Chronic long term complications can develop in both type 1 and type 2
diabetes. The main chronic complications of diabetes include:
• Diabetic cardiomyopathy and diabetic vasculopathy
• Diabetic nephropathy
• Diabetic retinopathy
• Diabetic neuropathy
Studies show that type 1 and type 2 diabetes complications arise from a
change in the balance of metabolites such as lipids, carbohydrates and
blood coagulation factors (Mard-Soltani et al. 2011). Chronic
complications may occur despite control of blood glucose. Studies show
that rapid tight control of blood sugar levels worsens diabetes
complication rather than improving the conditions (Taubes. 2008). Other
studies showed that the improved glucose control after 41 months did not
improve complications (Brinchmann-Hansen et al. 1988).
Hyperglycaemia is an important aetiological factor for diabetes
complications but studies show there are many other unknown factors
that may be involved based on the observation that complications
develop despite control of glucose. Type 2 diabetes is a chronic disease
with higher possibilities to develop chronic complications compared to
type 1 diabetes with a ten year shorter life expectancy.
The risk of cardiovascular complications including ischaemic heart
disease and stroke is increased by two to four times in type 2 diabetes
and there is a 20 fold increase in lower limb amputations (Williams
textbook of endocrinology. 2012 (12th ed,). Philadelphia:
Elsevier/Saunders. pp. 1371–1435.ISBN 978-1-4377-0324-5).
9
Type 2 diabetes is associated with the most frequent cause of non-
traumatic blindness and kidney failure. Other complications such as
sexual dysfunction and risk of infection are increased in type 2 diabetes
(Ripsin et al. 2009). Abnormal fat metabolism due to a lack of or
impaired insulin secretion and insulin resistance lead to hyperlipidemia
in diabetic patients. Increased levels of cholesterol and triglyceride are
metabolic complications of diabetes and increase the risk of organ
disease, particularly cardiovascular disorders. Elevated serum low
density lipoprotein ( LDL) is one of the most important side effect of
diabetes as a result of fat metabolism abnormalities and it is a target for
diabetes management to achieve an LDL level lower than 100mg/dl (2.6
mmol/l) (ADA, Standards of Medical Care in Diabetes - 2010. Diabetes
Care, 2010). Abnormal lipid metabolism, increased circulatory lipid
concentration and elevated deposition of lipids in the skeletal muscle are
widely displayed in patients with type 2 diabetes and insulin resistance
(McGarry, Banting lecture. Dysregulation of fatty acid metabolism in the
aetiology of type 2 diabetes. Diabetes 2001; 51: 7-18).
Hypertension is another complication of diabetes which affects
approximately 70% of diabetic patients (Rodrigo et al. 2007) and
directly increases the risk of cardiovascular and kidney complications
which need special attention and management.
Type 2 diabetes, insulin resistance, and untreated lack of insulin are
associated with high levels of serum uric acid (Cappuccio et al. 1993). It
has been shown that high serum uric acid increases the risk of Type 2
diabetes independent of the other risk factors such as obesity and
dyslipidemia (Dehghan et al. 2008). High serum uric acid can occur in
Type 1 diabetes and it is directly linked to renal damage, causes
increased morbidity and mortality (Rosolowsky et al. 2008).
10
1.4 Diagnosis Criteria
Based on WHO guidelines, diabetes is defined by recurrent or persistent
hyperglycaemia and is diagnosed by demonstrating one of the following
criteria: ("Definition, Diagnosis and Classification of Diabetes Mellitus
and its Complications" (PDF). World Health Organisation. 1999)
• Fasting Plasma Glucose level in greater than 125 mg/dl (7.0
mmol/l) from at least two independent measurements.
• Two-hour plasma glucose greater than 200 mg/dl (11.1 mmol/l)
after 75g oral glucose load (Oral Glucose Tolerance Test).
• Random blood glucose level greater than 200 mg/dl (11.1 mmol/l)
in presence of diabetes symptom including polydipsia, polyuria
and polyphagia.
• Haemoglobin A1C1 (Glycated Haemoglobin) greater than 6.5%.
Fasting blood glucose levels from 110 mg/dl to 125 mg/dl (6.1 to 6.9
mmol/l) and oral glucose tolerance test above 140 mg/dl (7.8 mmol/l) but
less than 200 mg/dl (11.1 mmol/l) are considered as impaired glucose
homeostasis and are indicative of pre-diabetic status.
1 Glycated hemoglobin or glycosylated hemoglobin (hemoglobin A1c, HbA1c, A1C, or
Hb1c; sometimes also HbA1c) is a form of hemoglobin that is measured primarily to
identify the average plasma glucose concentration over prolonged periods of time. It is
formed in a non-enzymaticglycation pathway by hemoglobin's exposure to plasma glucose.
Normal levels of glucose produce a normal amount of glycated hemoglobin. As the average
amount of plasma glucose increases, the fraction of glycated hemoglobin increases in a
predictable way. This serves as a marker for average blood glucose levels over the previous
months prior to the measurement. (Adapted from www.wikipedia.org ,
http://en.wikipedia.org/wiki/Glycated_hemoglobin )
11
The people at this stage are at a high risk to progress to full-blown
diabetes mellitus and diabetic complication including cardiovascular
diseases ("Definition, Diagnosis and Classification of Diabetes Mellitus
and its Complications" (PDF). World Health Organisation. 1999).
12
1.5 Insulin
1.5.1 Structure
Insulin is a polypeptide hormone with a molecular weight of 5808 Da
synthesized in the beta cells of the Islets of Langerhans in the pancreas
comprising two polypeptide chains, A with 21 amino acid residues, and
B with 30amino acid residues, which are linked by disulphide bridges.
Initially insulin is synthesised as pre-proinsulin with 110 amino acid
residues which is processed in the endoplasmic reticulum to proinsulin
containing 82 amino acid residues (Brunton LL, Chabner BA,
Knollmann BC: Goodman & Gilman's The Pharmacological Basis of
Therapeutics, 12th Edition:The McGraw-Hill Companies, 2011).
Proinsulin is comprised of chains A, B and the C-peptide. The C-peptide
domain has 31 amino acid residues and keeps the insulin molecule
inactive. Proinsulin will be transformed into active insulin by cleavage of
C-peptide via endopeptidases which cleave the bonds between lysine 64
and arginine 65, and between arginine 31 and 32 (Fig 1.4).
Conversion of proinsulin to active insulin is called maturation and is
carried out in cytoplasmic granules by prohormone convertases 2 and 3
and carboxy-peptidase H (Hutton 1994). Insulin has a variety of
biological effects in the body apart from maintaining glucose
homeostasis. Insulin is one of the potent anabolic hormone which
stimulates protein synthesis, lipogenesis, mitosis and cellular growth
(Tamas Fulop et al. 2003).
13
Fig 1.4. Synthesis and processing of insulin. The initial peptide, preproinsulin (110
amino acids) consists of a signal peptide (SP), B chain, C peptide, and A chain. The
SP is cleaved and S-S bonds form as the proinsulin folds. Two prohormone
convertases, PC1 and PC2, cleave proinsulin into insulin, and C peptide. Insulin and
C peptide are stored in granules and co-secreted in equimolar quantities. (Adopted
from: Brunton LL, Chabner BA, Knollmann BC: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 12th Edition:The McGraw-Hill Companies,
2011)
1.5.2 Secretion
Insulin secretion from beta cells happens in two phases, rapid triggered
and a slow release phase. The rapid triggered phase happens in response
to various stimulator including glucose, arginine, leucin, acetylcholin,
cholecyctokinin (CCK) (Cawston et al. 2010), glucagon-like peptide-1
(GLP-1), glucose-independent insulinotropic peptide (GIP) and
epinephrine via ß2 receptors (Layden et al. 2010). Glucose is the major
14
physiological stimulus of insulin release, entering beta pancreatic cells
via GLUT 2 transporters. Glucose is phosphorylated inside the cells by
glucokinase (hexokinase IV) to glucose-6 phosphate (G6P) which is a
rate-limiting step that controls glucose-regulated insulin secretion. G6P
enters the glycolytic pathway in cytosol, converted to pyruvate. Pyruvate
enters mitochondria to generate ATP through the citric acid cycle (Krebs
Cycle). Increased the ATP/ADP ratio and elevated ATP inhibit ATP-
sensitive potassium channels (Kir6.2, known as the sulfonylurea
receptor) on the membranes of beta cells. Reduced potassium efflux
leads to cell membrane depolarization which opens voltage- dependent
calcium channels and an influx of calcium into the cell. Increased
amounts of calcium stimulate exocytosis of insulin previously
synthesized granules via activation of phospholipase C (Fig 1.5)
(Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12th
Edition:The McGraw-Hill Companies, 2011 and Harrison's Principles of
internal medicine, 18th Edition, 2012).
Glucokinase acts as the glucose sensor in pancreatic beta cells, and has
unique features which distinguish it from other hexokinases and make it
function as a glucose sensor. It has lower affinity for glucose compared
to other hexokinases (higher Km of 10 mM, 100 times higher than other
hexokinases with Km of 0.1 mM), such that the activity increases with
rising concentrations of glucose and remains a half-maximal
concentration of glucose at 144 mg/dl (8 mmol/l). Other hexokinases are
inhibited by their products but glucokinase is not inhibited by G6P and
remains active to stimulate insulin release amid significant amounts of its
products (Matschinsky. 1996).
15
Fig 1.5. Mechanisms of glucose-stimulated insulin secretion and abnormalities in
diabetes. Glucose and other nutrients regulate insulin secretion by the pancreatic beta
cell. Glucose is transported by a glucose transporter GLUT 2; subsequent glucose
metabolism by the beta cell alters ion channel activity, leading to insulin secretion.
The SUR receptor is the binding site for some drugs that act as insulin secretagogues.
SUR, sulfonylurea receptor; ATP, adenosine triphosphate; ADP, adenosine
diphosphate, cAMP, cyclic adenosine monophosphate. IAPP, islet amyloid
polypeptide or amylin. (Adopted from: Harrison's Principles of internal medicine,
18th Edition)
1.5.3 Mechanism of Action of Insulin
Insulin elicits its biological effect through binding to insulin receptors on
cell plasma membranes expressed in all types of cells in the body. The
insulin receptor is a member of the tyrosine kinase receptor family and
has functional similarity to the insulin-like growth factor-1 (IGF-1)
receptor (Taniguchi et al. 2006). The insulin receptor is a transmembrane
16
receptor comprising two extracellular alpha subunits and two membrane-
spanning beta subunits attached by a disulfide link shaped a
heterotetramer with approximately molecular weight of 320 Da. Insulin
receptor subunits are encoded by INSR genes on chromosome 19
(Belfiore et al. 2009).
Alpha subunits act as ligand binding sites and beta subunits have
inherent tyrosine kinase activity. In the normal status of no ligand
binding, the alpha subunits inhibit the tyrosine kinase activity of beta
subunits. Binding of insulin to the alpha subunits changes the
conformation of the receptor and releases inhibition of alpha subunits,
leading to activation of the beta subunit tyrosine kinase. One beta subunit
transphosphorylates the other one, resulting in autophosphorylation. Beta
subunit activation initiates signalling cascade via phosphorylation of
intracellular proteins including insulin receptor substrate (IRS) and Src-
homology-2-containing protein (Shc) (Brunton LL, Chabner BA,
Knollmann BC: Goodman & Gilman's The Pharmacological Basis of
Therapeutics, 12th Edition:The McGraw-Hill Companies, 2011).
Phosphorylated IRS-1 protein binds to P85 regulatory subunit of
phosphatidylinositol 3-kinases (PI3Kinase) leading to activation of the
P110 catalytic subunit of the enzyme. Activated PI3Kinase
phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) in the
plasma membrane to phosphatidylinositol (3,4,5)-triphosphate (PIP3).
PIP3 activates protein kinase B (PKB or Akt) which facilitate GLUT 4
(glucose transporter 4) vesicle fusion to the plasma membrane resulting
in increased glucose uptake (Edward et al. 2005).
Activated protein kinase B inhibits glycogen synthesis by inhibiting
glycogen synthase kinase (GSK) through phosphorylation which leads to
inactivation of glycogen synthase (Xianjun Fang et al. 2000).
PI3Kinase activation triggers several downstream kinases via producing
17
PIP3 including Akt 2 (PKB), mammalian target of rapamycin (mTOR)
and protein kinase C (PKC) (Huang S and Czech MP. 2007). The
PI3Kinase I/Akt2 activation plays a major role in mediating insulin
action for glucose transport in skeletal muscle and adipose tissue and
glucose production regulation and liver cells (Zaid H et al. 2008).
Phosphorylation of beta subunits of insulin receptors activates Src-
homology-2-containing protein (Shc) and Growth factor receptor-bound
protein 2 (Grb 2) which leads to initiation of a protein kinase cascade
involving mitogen-activated protein kinase (MAPK) and extracellular-
signal-regulated kinases (ERKs) that mediate insulin actions for gene
transcription and cell growth (Fig 1.6) (Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 12th Edition:The McGraw-Hill
Companies, 2011).
MAPK cascade activation induces gene expression regulation, cell
growth and differentiation actions of insulin (Louise et al. 2008). MAPK
inhibitors inhibit insulin-stimulated glucose uptake without any
detectable effect on GLUT4 translocation (Sweeney et al. 1999). The
MAPK pathway is involved in intrinsic GLUT4 regulation, the
mechanism is still not well understood.
Activation of small GTP-binding proteins like TC-10 and Rac,
independent of PI3Kinase activation, is important for insulin action for
increasing glucose transport in peripheral cells as these proteins are
critical for vesicle trafficking (Kjoller et al. 1999 , Chiang et al. 2001).
Insulin signalling is terminated by degradation of receptor bound to
insulin and decreases the amount of insulin receptor on the plasma
membrane (Najjar. 2001). Dephosphorylation of tyrosine residues by
tyrosine phosphatases is another mechanism that leads to termination of
insulin signalling within the cells.
18
Fig 1.6. Pathways of insulin signalling. The binding of insulin to its plasma
membrane receptor activates a cascade of downstream signalling events. Insulin
binding activates the intrinsic tyrosine kinase activity of the receptor dimer, resulting
in the tyrosine phosphorylation (Y-P) of the receptor's beta subunits and a small
number of specific substrates (yellow shapes): the Insulin Receptor Substrate (IRS)
proteins, Gab-1 and SHC; within the membrane, a caveolar pool of insulin receptor
phosphorylates caveolin (Cav), APS, and Cbl. These tyrosine-phosphorylated
proteins interact with signalling cascades via SH2 and SH3 domains to mediate the
effects of insulin, with specific effects resulting from each pathway. In target tissues
such as skeletal muscle and adipocytes, a key event is the translocation of the Glut4
glucose transporter from intracellular vesicles to the plasma membrane; this
translocation is stimulated by both the caveolar and non-caveolar pathways. In the
non-caveolar pathway, the activation of PI3K is crucial, and PKB/Akt (anchored at
the membrane by PIP3) and/or an atypical form of PKC is involved. In the caveolar
pathway, caveolar protein flotillin localizes the signalling complex to the caveola; the
signalling pathway involves series of SH2 domain interactions that add the adaptor
protein CrkII, the guanine nucleotide exchange protein C3G, and small GTP-binding
protein, TC10. The pathways are inactivated by specific phosphoprotein
phosphatases (eg, PTB1B). In addition to the actions shown, insulin also stimulates
19
the plasma membrane Na+,K+-ATPase by a mechanism that is still being elucidated;
the result is an increase in pump activity and a net accumulation of K+ in the cell.
Abbreviations: APS, adaptor protein with PH and SH2 domains; CAP, Cbl associated
protein; CrkII, chicken tumor virus regulator of kinase II; GLUT4, glucose
transporter 4; Gab-1, Grb-2 associated binder; MAP kinase, mitogen-activated
protein kinase; PDK, phosphoinositide-dependent kinase; PI3kinase,
phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol trisphosphate; PKB,
protein kinase B (also called Akt); aPKC, atypical isoform of protein kinase C; Y,
tyrosine residue; Y-P, phosphorylated tyrosine residue. (Adapted from: Goodman &
Gilman's The Pharmacological Basis of Therapeutics, 12th Edition:The McGraw-Hill
Companies, 2011)
20
1.6 Glucose Transporters
Glucose is a vital source of energy and important metabolic substrate for
mammalian cells. Glucose transporters are a broad group of membrane
proteins which are responsible for glucose uptake and transport through
the plasma membrane of cells. In general glucose transporters comprise
two groups based on their function and structure including Na-dependent
glucose transporters (SGLT) (Wright. 2001) and facilitative Na -
independent sugar transporters (GLUT family) (Mueckler . 1994).
SGLTs are Na+/K+ ATPase pumps that facilitate glucose transport in the
small intestine (SGLT-1) and the proximal tubule of nephrons in kidney
(SGLT-1 and SGLT-2). SGLT-1 is a high affinity and SGLT-2 is a low
affinity Na-dependent glucose transporters (I. Stuart Wood et al. 2003).
GLUTs are tarnsmembrane proteins with 12 membrane-spanning helices
which facilitate diffusion of glucose and other sugars across the plasma
membrane. Binding of glucose to the extra-membrane site of GLUT
provokes a conformational change of the transporter and leads to release
of glucose from the other intra-membrane side of the transporter (Hruz et
al. 2001).
The GLUT family consists of three subclasses (Class I,II,III) with 13
members (Hruz et al. 2001, Joost et al. 2001). Each GLUT isoform
plays specific role in glucose uptake in different tissues and
physiological conditions. Substrate specificity and transporter kinetics
are different in each isoform which creates a specific role for each GLUT
isoform in sugar metabolism (Thorens. 1996). Class I GLUTs are well
defined and comprise GLUT 1 to GLUT4 (Table 1.1).
21
GLUT1 is a high affinity glucose transporter responsible for low-level
basal glucose uptake when the glucose concentration is low and is
widely expressed in erythrocytes and endothelial cells of vascular tissues
specially barrier tissues like the blood brain barrier (BBR). It is also
responsible for vitamin C uptake (Nelson et al. 2008).
GLUT2 is a very efficient glucose transporter with high capacity and low
affinity (Higher Km ≥ 10 mM), acts as a glucose sensor in pancreatic
beta cells and also carries glucosamine as well as glucose (Efrat. 1997).
Glut2 is also expressed in small intestine cells, proximal renal tubules
and hepatocytes. It plays an important role in transporting glucose in fed-
state from the intestinal lumen (Bell et al. 1990).
GLUT3 is a glucose transporter with very high affinity, five times greater
than other class I GLUTs, and is mainly expressed in neurons (Simpson
et al. 2008, Vannucci et al. 1997). GLUT1 transporter in the brain blood
barrier and GLUT3 with high affinity for glucose play a central role in
glucose uptake and metabolism in central nervous system at low glucose
concentration.
GLUT4 is the insulin-responsive glucose transporter found in adipose
tissue, skeletal muscle and heart which is responsible for the reduction of
postprandial plasma glucose rise in response to insulin (Rayner et al.
1994). Insulin increases glucose uptake immediately, 10-20 fold by
stimulating GLUT4 translocation in adipose tissue and skeletal muscle
(Shepherd et al. 1999). GLUT4 translocation in skeletal muscle can also
be increased by exercise, independent of insulin (Ploug et al. 1998).
22
Name Distribution Notes
GLUT1 Widely distributed in fetal tissues. In the adult, it is expressed
at highest levels in erythrocytes and also in the endothelial
cells of barrier tissues such as the blood–brain barrier.
However, it is responsible for the low-level of basal glucose
uptake required to sustain respiration in all cells.
Levels in cell membranes
are increased by reduced
glucose levels and
decreased by increased
glucose levels.
GLUT2 A bidirectional transporter, allowing glucose to flow in 2
directions. Is expressed by renal tubular cells, small intestinal
epithelial cells, liver cells and pancreatic beta cells.
Bidirectionality is required in liver cells to take up glucose
for glycolysis, and release glucose during gluconeogenesis.
In pancreatic beta cells, free flowing glucose is required so
that the intracellular environment of these cells can
accurately gauge the serum glucose levels. All three
monosaccharides (glucose, galactose and fructose) are
transported from the intestinal mucosal cell into the portal
circulation by GLUT2
Is a high-capacity and
low-affinity isoform.
There is some evidence
that GLUT 1 and 3 are
actually the functional
transporters in beta cells.
GLUT3 Expressed mostly in neurons (where it is believed to be the
main glucose transporter isoform), and in the placenta.
Is a high-affinity isoform,
allowing it to transport
even in times of low
glucose concentrations.
GLUT4 Found in adipose tissues and striated muscle (skeletal
muscle and cardiac muscle).
Is the insulin-regulated
glucose transporter.
Responsible for insulin-
regulated glucose
storage.
Table 1. Class I glucose transporters GLUT1-GLUT4. (Bell et al. 1990 and Thorens
et al. 2010)
As GLUT4 translocation in response to insulin is strictly dependent on
PI3Kinase activation and PI3Kinase inhibition completely prevents the
insulin activity from increasing glucose uptake via GLUT4, previous
studies show that inhibition of PI3Kinase by wortmannin has no effect
on GLUT4 translocation in skeletal muscle in response to exercise
(Brozinick et al. 1998).
23
GLUT5 is a specific transporter for fructose widely expressed at the
apical side of small intestine cells, and is responsible for transport of
dietary fructose (Burant et al. 1992). GLUT 5 is also found in many
other types of cells, such as skeletal muscle, adipose, brain and kidney.
GLUT 5 drew the attention in recent years researches because fructose is
an important component of human diets. Increasing of fructose
consumption in human diets in last decades raised a concern about the
contribution of fructose to development of metabolic disorders such insulin
resistance, diabetes and obesity as it was shown in many literatures that
fructose-fed rodents could develop some degree of insulin resistance and
diabetes. The peripheral cells uptake the fructose mainly via GLUT5 and
with less degree with other GLUTs and it will go into the metabolic
pathway of ATP production same as glucose but with an important
difference. Fructose unlike the glucose which converted to G6P via
hexokinase, converts to fructose 1 phosphate via fructokinase and end up to
glyceraldehyde. It bypasses the rate controlled glycolysis process which is
controlled by hexokinase negative feedback. Increasing G6P inhibits the
hexokinase and controls the rate of glycolysis and glucose metabolism
within the cell but conversion of fructose to fructose 1 phosphate does not
follow the same path. On the other hand, fructose could not increase the
insulin secretion from pancreatic beta cell same as glucose (Eliott et al.
2002).
Glucose transporters play a major role in glucose metabolism,
particularly GLUT4 as an insulin-stimulated rate limiting transporter.
Animal studies showed that suppression of GLUT4 expression leads to
insulin resistance and type 2diabetes (Stenbit et al. 1997).
24
1.7 Insulin Resistance
Insulin resistance refers to the condition in which responsiveness of cells
to insulin is decreased and leads to higher concentrations of insulin in the
blood required to maintain normoglycemic status. Insulin resistance is
one of the main pathogenesis pathways of type 2 diabetes and results in
defects in glucose uptake and metabolism. Insulin resistance creates an
imbalance in the insulin/glucagon ratio which leads to disruption of
cellular energy production via decrease of glycolysis. It also increases
liver glyconeogenesis which helps plasma glucose levels rise and
worsens the situation. In adipose tissue insulin malfunction increases
triglyceride hydrolysis (lipolysis) which leads to elevated free fatty acid
concentration in blood (Goodman & Gilman's The Pharmacological
Basis of Therapeutics, 12th Edition:The McGraw-Hill Companies, 2011).
Insulin resistance pathology is still not well characterised but previous
studies revealed some risk factors including obesity, race,family history,
hypertension, low level of high density lipoprotein (HDL), gestational
diabetes and birth weight more than 4 kg, are associated with the insulin
resistance condition (Jennifer Mayfield. 1998 and Type 2 diabetes: Risk
factors. Mayo Clinic. Retrieved 21 December 2011).
Among the risk factors, obesity and high fat intake especially saturated
fat have significant positive correlation with insulin resistance (J
Lovejoy et al. 1992, LH Storlien et al. 1991). The high level of free fatty
acid and triglyceride play an important role in the development of insulin
resistance ( Schinner et al. 2005). Some studies suggested that fructose
contributes to insulin resistance development as it is metabolised to
triglyceride in the liver, which increases triglyceride and free fatty acid
levels in the blood (Heather et al. 2005).
25
It is well established that feeding rats with fructose creates insulin
resistance and type 2 diabetes, which is used as a model for type 2
diabetes animal studies (Thorburn. 1989).
Insulin resistance increases insulin levels as a push to restore the
normoglycemic status but excess insulin stimulates fat storage and new
fat tissue formation which accelerates weight gain. This defective cycle
makes the condition worse. Unhealthy diet like fast food with high fat
and fructose content has been shown to play a fundamental role in
insulin resistance and metabolic syndrome conditions (Elvira et al.
2005).
It is well established that elevated plasma free fatty acid concentration
reduces insulin-stimulated glucose uptake, and vice versa, reduction in
plasma lipid concentration improves insulin activity and glucose uptake
in adipose tissue, skeletal muscle and liver (Moller. 2001). Elevated lipid
can create functional defects in insulin receptors and insulin receptor
substrates (IRS) which change the binding of ligands, insulin receptor
expression, the phosphorylation state of insulin receptor kinase domain
and the activity of receptor tyrosine kinases. High free fatty acid
concentrations also affect insulin signalling downstream via inhibition of
IRS-1 associated PI3Kinase activation in muscle and IRS-1 serine
phosphorylation which decreases tyrosine phosphorylation of IRS-1 and
leads to impaired IRS-1 activation (Yu C et al. 2002). It also inhibits
AKT (PKB) activation by increasing the amount of ceramide and
diacylglyerol in skeletal muscle (Chavez et al. 2003).
Elevated free fatty acid (FFA) concentration decreases insulin receptor
activity via activation and over-expression of protein kinase C (PKC)
(Greene et al. 2004). It has been demonstrated that activation and over-
expression of PKC lead to decrease of Akt (PKB) activity and insulin
receptor down-regulation which are the mechanisms involve in FFA-
26
induced insulin resistance (Ikeda et al. 2001). The insulin receptor gene
expression is regulated positively by binding HMGA1 (High-mobility
group protein 1) to its AT-rich sequences (C2 and E3) within the
promoter region. Transfection of cells with HMGA1 increases insulin
receptor expression which indicates the important role of HMGA1 in
insulin receptor regulation (Brunetti et al 2001). There is some evidence
that FFA-induced PKC activation leads to phosphorylation of HMGA1
which reduces its mobility and binding capability and result in insulin
receptor down regulation and insulin resistance (Samir et al. 2007).
There is strong evidence for connections between malonyl-CoA, acetyl-
CoA carboxylase (ACC1& ACC2) and insulin resistance. Injection of
ACC1 and ACC2 anti-sense inhibitors restored diet-induced hepatic
insulin resistance in rat (Savage et al. 2006). Acetyl-CoA carboxylase
(ACC) is an enzyme with two isoforms that converts acetyl-CoA to
malonyl-CoA and plays an important role in fatty acid synthesis (Tong L.
2005). ACC2 concentrations are much higher than ACC1 in oxidative
tissues like skeletal muscle and heart. Both ACC1 and ACC2 are widely
expressed in hepatic cells where both fatty acid oxidation and synthesis
are important. ACCs are regulated by allosteric activators such as citrate,
glutamate and dicarboxylic acids (Martin et al. 1962 and Boone AN et al.
2000) and phosphorylating inhibitor, AMP-activated protein kinase
(AMPK) which leads to deactivation of ACCs (Park et al. 2002).
Activation of AMPK in muscle inhibits ACC2 and increases fatty acid
and glucose oxidation and decreases levels of malonyl-CoA. It also
increases the GLUT4 content in membrane and up-regulates the GLUT 4
expression (Koistinen et al. 2003). AMPK as a negative regulator of
ACC activity is a major target for treatment of type 2 diabetes and
insulin resistance (Ruderman et al. 2004).
27
Glucagon and catecholamines inhibit ACC via AMPK activation whereas
glucose and insulin increase ACC activity by dephosphorylation via
phosphatase activation (Kim et al. 1997, Witters et al. 1988).
Regulation of malonyl- CoA and fatty acid metabolism are playing an
important role in beta pancreas cells and insulin secretion as well as
adipose tissue and skeletal muscle. There is a notion that changes in
malonyl-CoA, fatty acid synthesis and oxidation regulation could be
involved in obesity and insulin resistance as the fundamental
pathogenesis of type 2 diabetes (Prentki et al. 1996).
Abnormalities in energy homeostasis regulation is a key point in
metabolic syndrome that leads to a variety of conditions and diseases
like diabetes, insulin resistance, cancer and cardiovascular diseases. The
insulin/IGF (insulin-like growth factor) pathway is a major regulator of
energy homeostasis by controlling glucose and lipid metabolism and
regulation of their homeostasis (Biddinger et al. 2006).
Forkhead transcription factor (FOXO) is an important mediator for the
metabolic effects of the insulin/IGF pathway. FOXO family has three
main variants encoded by three genes (FOXO1,3 and 6). FOXO1 is the
main controller of glucose homeostasis in peripheral tissues and
pancreatic beta cells (Accili et al. 2004).
FOXO1 is a critical mediator of insulin signalling as over activation and
expression of FOXO1 in hepatic and pancreatic beta cells causes hepatic
insulin resistance and apoptosis of pancreatic beta cells (beta cell loss)
which are reversed by reduction of FOXO1 function (Kitamura et al.
2002, Nakae et al. 2002).
The mTOR (mammalian target of rapamycin) pathway is another
28
important mediator of insulin/IGF pathway, it directly controls Akt
(Protein Kinase B) function via its phosphorylation. It also suggests
dysregulation of mTOR signalling may have a role in pathogenesis of
metabolic syndrome and insulin resistance (Sarbassov et al. 2005).
PPAR-γ (Peroxisome Proliferator-Activated Receptors Gamma) is a
nuclear protein and plays an important role in insulin sensitivity and
glucose homeostasis (Picard et al. 2002). Ligand binding to PPAR-γ
creates conformational changes and stabilizes its interaction with RXR
(Retinoid X Receptor). PPAR-γ/RXR interaction stimulates gene
expression by recruiting a set of coactivators to the promoter region of
the targeted gene (Berger et al. 2002). In-vivo and in-vitro studies
showed that PPAR-γ activation increases insulin sensitivity and enhances
insulin-induced glucose uptake in adipocytes (Norris et al. 2003 and
Nugent et al. 2001). It also has been shown that PPARγ co-activator-1
(PGC-1) has a crucial role in glucose homeostasis via regulation of
GLUT4 gene expression in which PGC-1 strongly induces endogenous
GLUT4 gene expression (Michael et al. 2001).
Chronic low-grade inflammatory responses are associated with insulin
resistance in adipose tissue (Ruan et al. 2003, Hotamisligil et al. 2003).
It was well demonstrated that macrophage content and pro-inflammatory
gene activation are increased in adipose tissue in obesity and insulin
resistance status. There is strong evidence that activation of the TNF-α
pathway, as an inflammatory cytokine response desensitizes insulin
signalling and creates insulin resistance (Hotamisligil et al. 1995,
Hotamisligil et al. 1994, Hotamisligil et al. 1993, Uysal et al. 1998).
TNF-α pathway activation down-regulates GLUT-4 expressions by
phosphorylation of serine residue IRS-1 which inactivate IRS-1 and
29
insulin signalling (Uysal et al. 1997). Several studies have shown that
PPARγ agonists inhibit secretion of TNF- α, macrophage activation and
also reduce the TNF- α -induced inhibition of insulin signalling in
adipose tissue (Ricote et al. 1998, Peraldi et al. 1997, Ruan et al. 2003).
Mitochondrial dysfunction can cause insulin resistance and diabetes,
since mitochondria are the main organelle in cells which maintain energy
homeostasis. Mitochondria density is reduced by 38% in the muscle of
young insulin-resistant offspring of type 2 diabetes parents (Morino et al.
2005).
Other regulatory hormones secreted from adipocytes, known as
adipokines, are involved in glucose homeostasis modulation including
adiponectin, resistin and leptin. Leptin is a 16 KDa protein, which
regulates appetite and energy intake (Brennan et al. 2006). Leptin has
both central and peripheral mode of actions. It was shown that interacts
with six types receptors encoded by LEPR (Wang et al. 1996). There is a
strong connection between leptin and obesity. Leptin inhibits the
neuropeptide Y secretes neurons in arcuate nucleus in the brain which
controls the satiety feeling (Baicy et al. 2007). Apart from leptin central
activity in connection with hypothalamus, leptin's receptors are
expressed in wide types of peripheral cells. It has been shown that leptin
plays a role in cellular energy expenditure modulation via interacting
with metabolism regulatory hormones and other energy regulation
mechanisms (Margetic et al. 2002). People with leptin receptor
mutations or leptin deficiency suffer from obesity (Montague et al. 1997,
Farooqi et al. 1999). As obesity is a well established risk of insulin
resistance, leptin may be involved in insulin resistance.
Adiponectin is another important modulator which increases insulin
sensitivity and glucose uptake in both adipose tissue and muscles and
30
reduces gluconeogenesis and glucose output in hepatic cells. It was also
shown that expression of adiponectin decreases in obese humans
(Stumvoll et al. 2002). Opposite to adiponectin, resistin increases
gluconeogenesis and hepatic glucose output and decreases insulin
sensitivity and insulin-induced glucose uptake in both adipose and
muscule tissues (Moon et al. 2003, Pravenec et al. 2003). There is a
strong belief that adipokines may contribute to insulin resistance and
pathogenesis of type 2 diabetes. They also attracted much research
interest in recent years, not only for understanding the molecular
pathology of insulin resistance, but also for new therapeutic approaches.
Other genomic regulators in insulin secretion and signalling are
microRNAs (miRNA) which are recently attracting many researches in
order to identify their role in pathogenesis of diabetes. MicroRNAs are
short single-stranded non protein coding gene products with length of
19-23 nucleotides which post-transcriptionally regulate the expression of
genes by interacting with specific mRNAs (Bartel. 2004, Lagos-
Quintana et al. 2001, Fabian et al. 2010). MicroRNAs interaction with
targeted mRNAs lead to mRNA degradation or inhibition of mRNA
translation which results in negative regulation (Tranzer et al. 2006,
Ying et al. 2006). It was established that microRNAs are directly
involved in glucose homeostasis, insulin secretion and insulin sensitivity,
as shown in Table 1.2. A recent study showed that a plasma signature of
five miRNAs (miR-15a,miR-29b, miR-126, miR-223, and miR-28–3p) is
closely connected with a high likelihood of developing type 2 diabetes
(Zampetaki et al. 2010). These miRNAs are indicators of early type 2
diabetes and responsible for its progression (Regazzi. 2010). There is a
hypothesis that miRNA may transmit from one tissue to another and act
at a distance from their sites of biogenesis (Dinger et al. 2008). There is
31
supportive evidence for this hypothesis as it was shown that miR-150 is
taken up by target endothelial cells from the bloodstream (Zhang et al.
2010). It suggests that microRNAs may be involved in insulin resistance
and diabetes pathogenesis at the genomic level, which needs further
detailed investigation. miRNAs could be targets for therapeutic purposes
and developing new medications.
Biological Process Specific MicroRNAs
ngn3-independent endocrine pancreas
regeneration
miR-15a, miR-15b, miR-16 and miR-195
Regulates insulin expression miR-30d, miR-375, miR-124a2
Regulates insulin secretion miR-375, miR-9, miR-124a2
Regulates glucose-stimulated insulin
secretion (GSIS)
miR-369-5p, miR-130a, miR-27a, miR-410, miR-200a,
miR-337, miR-532, miR-320, miR-192 and miR-379,
miR-375, miR-124a2
Regulates adipocyte differentiation miR-143, miR-27b, miR-130, miR-519d
Regulates insulin sensitivity miR-103, miR-107, miR-29, miR-320, mmu-mir-183-
96-182 (cluster)
Table 1.2. Summary of key biological processes and miRNAs (Adopted from:
Michael D.Williams et al. 2012)
There is a strong genetic links between diabetes type 1 and many genes.
It was shown that there is a strong link between histocompatibility gene
loci on chromosome 6 which encodes MHC class II (HLA) proteins and
diabetes type I (Bluestone et al. 2010). It was shown in the same study
that some variant of HLA gene including DRB1 (0401, 0402, 0405),
DQA 0301 and DQB1(0302 and 0201) increase the risk of type 1
diabetes. However, these variants are also found in the general
population, and only about 5 percent of individuals with the gene
variants develop type 1 diabetes. HLA variations account for
approximately 40 percent of the genetic risk for the condition. Other
HLA variations appear to be protective against the disease.
32
1.8 Diabetes management and available therapeutics
Diabetes management has two main categories, insulin therapy and anti-
diabetic medications. Insulin replacement is the main treatment of type 1
diabetes in which the insulin producing capability of beta cells has been
lost. Anti-diabetic medication has got no, or a minimal place in the
treatment of type 1 diabetes. Insulin therapy in type 1 diabetes is
necessary in order to maintain normoglycemic status in the long term,
but increases the risk of small blood vessel disease and may promote
cardiovascular complications of diabetes (Sugimoto et al. 2003,
Mudaliar . 2009). Insulin therapy remains as a last resort for type 2
diabetes particularly when other therapies with anti-diabetic medications
fail to control blood glucose level.
The goals for treatment of diabetes based on the American Diabetes
Association (ADA) recommendations are:
• HbA1c lower than 7%
• Peripheral plasma glucose level between 70 to 130 mg/dl (3.9 to
7.2 mmol/l)
• Peak postprandial capillary plasma glucose (OGTT) lower than
180 mg/dl(10 mmol/l)
• Low density lipoprotein (LDL) lower than 100 mg/dl (2.6 mmol/l)
• High density lipoprotein (HDL) greater than 40mg/dl (1 mmol/l)
in men and 50 mg/dl (1.3 mmol/l) in women
• Triglyceride lower than 150 mg/dl (1.7 mmol/l)
The aims of treatment for both type 1 and 2 diabetes are to allow patients
33
to achieve a normal lifestyle, to eliminate hyperglycaemic symptoms,
and to reduce long term complications of diabetes, especially
cardiovascular complication.
Anti-diabetic medications are divided into four main categories as
sensitizers, secretagogues, alpha glucosidase inhibitors and peptide
analogues.
Sensitizers: Biguanides, like metformin exert their anti-hyperglycaemic
action by suppressing gluconeogenesis in liver and enhance glucose
uptake via AMP-activated protein kinase (AMPK) activation
(Kirpichnikov et al. 2002, Musi et al. 2002). Thiazolidinediones like
troglitazone, pioglitazone and rosiglitazone are another member of this
category. Thiazolidinediones are PPAR-γ agonists, which increase
insulin sensitivity.
Secretagogues: Sulfonylureas like tolbutamide, glipizide, gliclazide,
glibenclamide and Meglitinides know as non-sulfonylurea secretagogus
or short-acting secretagogus like repaglinide and nateglinide are
members of this family. This family increases insulin secretion by
inhibiting ATP-sensitive potassium channels in beta cells which leads to
beta cell membrane depolarization and an influx of calcium and
exoytosis of insulin vesicles.
Alpha Glucosidase Inhibitors: Acarbose and miglitol are members of
this group, which reduce glucose absorption by slowing down starch
digestion. This group technically is not categorized as hypoglycaemic
agents but they help glucose to enter the blood stream from the intestine
slowly, which can be matched effectively with an impaired insulin
response.
34
Peptide Analogues: Incretins including glucagon-like peptide1 (GLP-1)
and glucose-dependent insulinotropic peptide (GIP) are secreted after
meals and stimulate insulin secretion. They have a very short half life up
to few minutes. GLP-1 is effective in type 2diabetes for stimulating
insulin secretion whereas GIP is not. GLP-1 agonists including exenatide
and liraglutide are effective in type 2 diabetes patients after a meal by
stimulating insulin release, inhibiting glucagon and delaying gastric
emptying (Brunton LL, Chabner BA, Knollmann BC: Goodman &
Gilman's The Pharmacological Basis of Therapeutics, 12th Edition:The
McGraw-Hill Companies, 2011). Incretins are rapidly inactivated by
dipeptidyl peptidase-4 (DPP-4). Recently a new category of anti-diabetic
medication has been developed as DPP-4 inhibitors like sitagliptin and
linagliptin. DPP-4 inhibitors reduce HbA1C (Fig 1.7) (Amori et al.
2007). All the available medications as a treatment regiment for diabetes
work on utilising insulin secretion or increasing its effect on peripheral
cells to reduce the blood glucose level but not targeting the fundamental
metabolic disorder of diabetes.
Fig 1.7. Incretins activity and action of DPP-4 inhibitors. DPP-4 normally inactivates
GLP-1. DPP-4 inhibitors block DPP-4 which in turn leaves GLP-1 active.
35
1.9 Fenugreek (4-Hydroxyisoleucine)
Trigonella foenum-graecum L. (Legominosae) known as Fenugreek (Fig
1.8) is one of the oldest medicinal plants and is native to southeastern
Europe, northern Africa and western Asia. Fenugreek seeds often have a
spicy and pungent aroma and bitter taste. Fenugreek seeds contain many
active compounds such as iron, vitamin A, B, C, phosphates, flavonoids,
saponins, alkaloids such as trigonelline and amino acids (Fenugreek seed
bioactive compositions and methods for extracting same. United States
Patent 7338675). The leaves and seeds have a long record of historical
usage in many countries where it was grown for medicinal purposes.
Applications of fenugreek were documented in ancient Egypt as incense
for embalming mummies. Fenugreek is used as a supplement in wheat
and maize flour for bread-making in modern Egypt (Morcos et al. 1981).
In traditional Chinese medicine, fenugreek seeds are consumed as a
tonic, as well as a remedy for weakness and oedema of legs (Basch et al.
2003). Fenugreek is commonly used as a condiment in India and used as
a lactation stimulant as well (Yoshikawa et al. 1997). In Iranian
traditional medicine the seeds have been used as a blood sugar lowering
herb (Amin Gh. Popular medicinal plants of Iran. Tehran: Vice-
choncellor of Research of Tehran University of Medical Sciences, 1991,
pp: 101 – 2).
The possible hypoglycemic properties of oral fenugreek seed powder
have been suggested by the results of preliminary animal and human
trials. The studies showed that defatted seeds are associated with the
hypoglycemic effects of fenugreek seeds. These effects have not been
observed in studies of lipid extracts (Basch et al. 2003).
36
Fig 1.8. Trigonella foenum-graecum (Fenugreek) (Prof. Dr. Otto Wilhelm Thomé
Flora von Deutschland, Österreich und der Schweiz 1885, Gera, Germany)
Administration of 100 g of defatted fenugreek seed powder to people
with type 1 diabetes in 10 days showed significant amelioration in
diabetes associated symptoms (Sharma et al. 1990). Another study on
patients with type 2 diabetes showed that fenugreek seed powder
administration for 6 weeks reduced blood glucose and cholesterols (Abu
Saleh et al. 2006). Some studies conducted on fenugreek have focused
on investigating the effect of a specific sub-fractions of the fenugreek
seeds containing a unique amino acid known as 4-hydroxyisoleucine in
animals and humans with diabetes or lipid disorder (Fenugreek seed
bioactive compositions and methods for extracting same. United States
Patent 7338675).
The previous works on 4-hydroxyisoleucine proved that it acts as a
potent blood sugar lowering and anti-dyslipidemic agent (Broca et al.
2004, Sauvaire et al. 1998, Al-Habori et al. 1998). 4-hydroxyisoleucine
is a branch amino acid which has been extracted from fenugreek seeds
and it has not been found in any mammalian tissues (Fig 1.9 &Table
1.3), or in other plants.
37
A B
Fig 1.9. A: (2R,3R,4S) 4-Hydroxyisoleucine. B: (2S,3R,4S) 4-Hydroxyisoleucine.
(Adapted from Toronto Research Chemicals Inc, http://www.trc-canada.com)
CAS Number 781658-23-9
IUPAC Name 2-amino-4-hydroxy-3-
methylpentanoic acid
Synonym 2-Amino-2,3,5-trideoxy-3-methyl-D-
xylonic Acid
Major Isomer (2S,3R,4S)-4-Hydroxyisoleucine
Minor Isomer (2R,3R,4S)-4-Hydroxyisoleucine
Molecular
Formula
C6H13NO3
Molecular Weight 147.17g/mol
Melting Point >203°C
Table 1.3. 4-Hydroxyisoleucine chemical specifications.
The studies have confirmed the presence of 4-hydroxyisoleucine in
fenugreek seeds in two diastero-isomers: the major one is the (2S, 3R,
4S) configuration, representing about 90% of the total content of 4-
hydroxyisoleucine in the fenugreek seeds extract, and the minor one is
the (2R, 3R, 4S) configuration (Alcock et al. 1989). The major isomer is
presently interesting with respect to experimental evidence indicating its
ability to stimulate glucose-induced insulin secretion in micromolar
38
concentrations (Sauvaire et al. 1998).
Some studies claimed that the natural analogue of 4- hydroxyisoleucine
is more effective as an anti-diabetic agent than a synthetic version. There
is, therefore, a suggestion that the therapeutic effects of 4-
hydroxyisoleucine are best obtained from extracts of the fenugreek seeds
(Fenugreek seed bioactive compositions and methods for extracting
same. United States Patent 7338675). But another study has shown
synthetic and natural 4-hydroxyisoleucine are same in hypoglycemic
properties. (Rolland-Fulcrand et al. 2004) 4-hydroxyisoleucine is safe to
be consumed by human without geneotoxicity as tested based on FDA
(US Food and Drug Administration) protocols and recommended 4-
hydroxyisoleucine for food ingredients (Flammang et al. 2004).
39
1.10 Isoleucine
Isoleucine (Ile) is an essential α-amino acid that cannot be synthesized by
humans. It is hydrophobic due its hydrocarbon side chains. Isoleucine
can have four possible stereoisomers but in nature it exists only as one
enantiomeric form, (2S,3S)-2-amino-3-methylpentanoic acid. Isoleucine
is a glucogenic amino acid involved in the metabolism of carbohydrate
which can be converted to succinyl-CoA and acetyl-CoA and fed to the
TCA cycle (Tricarboxylic Acid Cycle) (Fig 1.10) (Table 1.4) (Tappy et
al. 1992).
Fig 1.10. L-Isoleucine. (Adapted from Sigma Aldrich, http://www.sigmaaldrich.com)
CAS Number 73325
IUPAC Name (2S,3S)-2-Amino-3-methylpentanoic
acid
Molecular
Formula
C6H13NO2
Molecular Weight 131.17g/mol
Table4. L-Isoleucine chemical specification.
40
In-vivo study shows that isoleucine has hypoglycaemic properties and
can lower the blood glucose level and its hypoglycaemic effect reduces
when its blood level concentration reaches saturation (Doi et al. 2007).
Both Isoleucine and 4-hydroxyisoleucine with very close molecular
structure has got anti-diabetic effects (Ikehara et al. 2008) but
isoleucine's hypoglycaemic effect is not dependent on the concentration
of glucose as 4-hydroxyisoleucine glucose lowering effect is only
observed in high glucose concentration. It seems the extra OH group in
4-hydroxyisoleucine creates some unique anti-diabetic properties which
are not seen in isoleucine.
41
1.11 Background (4-Hydroxyisoleucine)
Diabetes is a progressive multi-factorial metabolic disease with serious
short and long term complications affecting different organs in the body
with high increasing prevalence in the world. Understanding the basis of
pathogenesis of diabetes specially type 2 diabetes which includes more
than 90% of diabetes cases worldwide and finding effective remedies are
very important as perspective of public health. It puts any potential
substances with anti-diabetic effect in the centre of attention in point of
view of researchers in relative fields. Fenugreek seeds as mentioned in
the last chapter has got a strong historical track in traditional medicine of
several cultures in Asia and middle-east for treatment of people with
diabetes symptoms.
Sharma in 1990 fed type 1 diabetes patients with 100 g of defatted
fenugreek seed powder per day divided in two doses for 10 days and
results showed a significant decrease in fasting blood glucose level, 54%
reduction in 24 hour urinary glucose excretion and improved glucose
tolerance test. It also indicated that serum total cholesterol, LDL, VLDL
and triglyceride have been significantly modified after 10 days without
any changes in HDL level (Sharma et al. 1990).
Another study of using 25 g twice a day of fenugreek seed powder in 30
type 2 diabetes patients with hyperlipidemia for 6 weeks showed that
serum total cholesterol, LDL and triglyceride have been reduced
significantly compared to control group but the HDL level remained
unchanged (Abu Saleh et al. 2006).
Gupta et al tested the effect of 1 gm/day hydroalcoholic extract of
42
fenugreek seeds on 25 newly diagnosed type 2 patients which showed a
reduction in blood glucose level, an increase in insulin sensitivity using
HOMA-model1 insulin resistance and decrease in triglyceride after two
months treatment. In this study they observed an increase in HDL level
in the treatment group as well (Gupta et al. 2001).
Analava et al showed that anti-diabetic and anti-dysplipidemic effect of
fenugreek seed powder is dose dependent and consuming more than 75
g/day can not produce significant differences in its effect on blood
glucose level. The blood glucose lowering effect between 75 g and 100 g
a day remained unchanged (Analava et al. 2006).
It was important question to determine what is responsible for anti-
diabetic and anti-dyslipidemic effect of fenugreek seed powder or extract
which well established in several studies. In 1979, Hardman detected 4-
hydroxyisoleucine and its isomers in fenugreek seed extract (Hardman et
al. 1979). This unique amino acid which only existed in fenugreek seed
and has been never found in any mammalian tissues or other plants up to
now, drew the attention of researchers. It could be responsible for such
effect of fenugreek seed.
There is no strong evidence that shows fenugreek leaves extract
producing such effects. It strengthened the idea that these effects may be
1 Homeostatic model assessment (HOMA) has been described by Matthews et al in 1985 and widely
employed in clinical research to assess insulin sensitivity. HOMA is calculated as the product of the
fasting values of glucose (expressed as mg/dL) times insulin (expressed as µU/mL) is divided by a
constant (405): HOMA= Glucose x Insulin /405. The constant 405 should be replaced by 22.5 if
glucose is expressed in S.I. units. The HOMA calculation compensates for fasting hyperglycaemia.
HOMA and insulin values increase in the insulin-resistant patient while the Glucose/Insulin ratio
decreases. The HOMA value correlates well with clamp techniques and has been frequently used to
assess changes in insulin sensitivity after treatment.( Matthews et al. 1985)
43
related to 4-hydroxyisoleucine as it is not that much inside the leaves.
In 1998, Sauvaire Y et al characterised 4-hydroxyisoleucine from
fenugreek seeds and showed its anti-diabetic effect. Their study of
administration of 4-hydroxyisoleucine directly to rat pancreas revealed
insuliotropic effect of 4-hydroxyisoleucine in the concentration range
100 micromol/l to 1mmol/l. They also determined that 4-
hydroxyisoleucine effect on stimulating insulin secretion from islets of
Langerhans in pancreas strictly dependent on glucose concentration. 4-
hydroxyisoleucine insulin stimulatory is ineffective in 3 and 5 mmol/l
concentrations of glucose. Insulin secretion is induced by 4-
hydroxyisoleucine at 6.6 to 16.7 mmol/l of glucose concentrations
(Sauvaire et al. 1998).
Broca and his colleagues in 1999 demonstrated that administration of
50mg/kg/day of 4-hydroxyisoleucine to type 2 diabetes rat model
significantly reduces basal hyperglycaemia and basal insulinemia. It also
improved glucose tolerance test. They showed that 4-hydroxyisoleucine
with concentration of 200 micromol/l induces insulin release from type 2
diabetic rat-isolated islets in exposure to high glucose concentration of
16.7 mmol/l (Broca et al. 1999). In 2000, Broca et al observed that the
insuliotropic property of 4-hydroxyisoleucine only seen in the
micromolar range of major isomer (2S,3R,4S)-4-hydroxyisoleucine
(Broca et al. 2000). In 2004, Broca investigated extra-pancreatic effect of
chronic 4-hydroxyisoleucine treatment in insulin resistant Zucker fa/fa
rats and sucrose-lipid diet fed rats.
Study results demonstrated that 4-hydroxyisoleucine increases peripheral
glucose utilization and decrease insulinemia in sucrose-lipid diet fed rats
and reduces hepatic glucose production and progression of
44
hyperinsulinemia in insulin resistant Zucker fa/fa rats. He also showed
that a single injection of 18 mg/kg of 4-hydroxyisoleucine increased
PI3Kinase activity associated with IRS-1 in both muscle and liver same
as insulin. Combining insulin and 4-hydroxyisoleucine did not increase
PI3Kinase activity further. This study indicated that 4-hydroxyisoleucine
has got insulin sensitizing property as well and increases insulin effect
on utilizing peripheral glucose via interaction with early signalling of
insulin (Broca et al. 2004).
Fenugreek seed extract containing minimum 40% 4-hydroxyisoleucine
tested for genetic toxicity by Flammang et al from Abbott laboratories in
America based on recommended US Food and Drug Administration
(FDA) genetic toxicity evaluation protocols using a reverse mutation
assay, mouse lymphoma forward mutation assay and mouse
micronucleus assay. The negative assays results demonstrated that
fenugreek seed extract and 4-hydroxyisoleucine are not genotoxic and
safe to be consumed by human (Flammang et al. 2004).
There are some controversies in studies of 4-hydroxyisoleucine on
skeletal muscle glycogen re-synthesis. Ruby et al evaluated oral
fenugreek seed extract containing 4-hydroxyisoleucine with glucose
drink and dextrose on trained male cyclist post-exercise skeletal muscle
glycogen resynthesis after 90 minutes intense ride. They found that 4-
hydroxyisoleucine plus glucose increases the rate of glycogen
resynthesis by 63% compared to glucose alone. Results of dextrose
showed the glycogen re-synthesis significantly enhanced by 4-
hydroxyisoleucine compared to dextrose alone (Ruby et al. 2005). They
repeated the experiments few years later by changing the protocol from
90 minutes intense exercise to 5 hours at 50% of peak cycling powder
45
and they reported that 4-hydroxyisoleucine does not improve glycogen
resynthesis. They suggested that the difference between the results of the
current study with previous work is due to differences in experimental
protocol. Interestingly, comparison both study findings shows that 4-
hydroxyisoleucine is effective in enhancing glycogen re-synthesis in
high intensity muscle activity rather than long term low intensity muscle
exercise (Slivka et al. 2008).
It is suggested that 4-hydroxyisoleucine supplement has preventive
efficacy on obesity induced by high fat diet as shown in animal study
that 4-hydroxyisoleucine reduces body weight gain and plasma
triglyceride gain in obese fat induced by high fat diet and oil
administration treated with fenugreek seed extract containing 4-
hydroxyisoleucine (Handa et al. 2005). It has also been shown that 4-
hydroxyisoleucine significantly decreases plasma triglyceride level by
33%, serum total cholesterol by 22% and plasma free fatty acid by 14%
in dyslipidemic hamster (Narender et al. 2006).
Haeri et al in 2009 showed that long term treatment of fructose-fed rats
(insulin resistance model) and streptozotocin-induced diabetes type 2 rats
with 50 mg/kg/day 4-hydroxyisoleucine for 8 weeks restored
significantly elevated liver damage markers including aspartate
transaminase (AST) and alanine transaminase (ALT) to near control
group values in fructose-fed rats but not in streptozotocin-induced
diabetes type 2 rats. They also showed long term treatment with 4-
hydroxyisoleucine is well tolerated in both models and 4-
hydroxyisoleucine increased HDL level in streptozotocin-induced
diabetes type 2 rats by 31% compared to control group (Haeri et
al.2009).
46
Singh and his colleagues in 2010 published a study using 50 mg/kg/day
4-hydroxyisoleucine isolated from fenugreek seeds on C57BL/KsJ-db/db
mice. They observed that 4-hydroxyisoleucine significantly decreases
elevated blood glucose level, total cholesterol, LDL and triglyceride in
well-characterised type 2 diabetes mice model. They also noticed a
significant increase in HDL level. It was suggested in this study that 4-
hydroxyisoleucine suppresses progression of type 2 diabetes by
increasing insulin sensitivity and peripheral glucose uptake (Singh et
al. 2010).
A recent published study by Jaiswal N et al on rat L6-GLUT4myc
skeletal muscle cells treated with 5 and 25 µ/ml 4-hydroxyisoleucine for
16 hours showed that 4-hydroxyisoleucine significantly and dose
dependently increases glucose uptake and time dependently GLUT4
translocation from plasma to the membrane of the cells after adding 100
nM insulin for 20 minutes. It was observed that incubation cells with 4-
hydroxyisoleucine and insulin together for 16 hours has not significant
effect on insulin-induced GLUT4 translocation compared to insulin
alone as a control. Treatment with 4-hydroxyisoleucine did not change
the cellular contents of GLUT1 and GLUT4 which prove that its effect
on glucose uptake is due to modification of signalling leads to GLUT4
translocation. 4-hydroxyisoleucine-induced GLUT4 translocation
completely blocked by adding 1 µ/ml of cycloheximide (CHX) which is
a potent protein synthesis inhibitor. Cycloheximide (CHX) can not
inhibit GLUT4 translocation alone and it was suggested that the increase
in GLUT4 translocation by 4-hydroxyisoleucine requires synthesis of
new proteins. It is also shown that adding wortmannin, a potent inhibitor
of PI 3Kinase inhibits the 4-hydroxyisoleucine-induced GLUT4
translocation, which identifies the key role of PI 3Kinase in 4-
47
hydroxyisoleucine effect. 4-hydroxyisoleucine also significantly
increased Akt (PKB) basal phosphorylation same as insulin without
changing the mRNA expression of Akt (PKB), IRS-1 and GLUT4 in
genetic level. This work confirms the effect of 4-hydroxyisoleucine on
utilizing peripheral glucose and increasing insulin sensitivity via its
signalling pathway as demonstrated in previous studies (Jaiswal et
al. 2012).
48
1.12 Background (Isoleucine)
Ikehara and his colleagues published an animal study in 2008 about the
effect of L-isoleucine in diabetes. They treated glucose intolerant high-
fat diet (HFD) mice, severe type 2 diabetes db/db mice and normal mice
with oral isoleucine at a dose of 30-300 mg/kg for 6 weeks. The results
showed that isoleucine significantly and dose-dependently decreases
blood glucose level in all groups including normal mice. It also
significantly increases blood insulin levels only in high-fat diet (HFD)
mice but not in other groups which suggests different mechanisms
involved in hypoglycemic property of isoleucine apart from its
insulinotropic activity on HFD mice. Insulin release dramatically
reduced in high-fat diet (HFD) mice treated chronically with isoleucine
after an oral glucose challenge without any changes in glucose tolerance
curve which indicates insulin-sensitizing property of isoleucine. This
study determined that both insulin secretion dependent and independent
mechanisms are involved, but based on results, insulin secretion
independent mechanism is playing a major role in its hypoglycaemic
effect specially in normal and sever diabetic conditions (Ikehara et al.
2008).
There is evidence the L-isoleucine increases insulin release from
pancreatic beta cells when added together with L-glutamine (Sener et al.
1981). In-vitro study on muscle C2C12 myotubes in the absence of
insulin showed that isoleucine with concentration 1 mM significantly
increased glucose consumption by 16.8% compared to control and l mM
leucine. Isoleucine did not increase glycogen synthesis compared to
leucine which significantly increased intra cellular glycogen synthesis.
The study also suggested that isoleucine stimulates insulin-independent
49
glucose uptake mediated by activating PI3Kinase and independent of
activation of mammalian target of rapamycin (mTOR) like leucine which
is a potent activator of mTOR (Doi et al. 2003, Zhang et al. 2007).
Another study by Doi et al showed that oral administration of 1.35 g/kg
L-isoleucine to fasted rats for 18 hours significantly reduced blood
glucose level compared to 1.35 g/kg L-leucine and control. L-leucine did
not change the blood glucose level. Isoleucine increased skeletal muscle
glucose uptake by 73% without changing glycogen synthesis and AMKP
alpha-1( AMP-activated protein kinase). In contrary leucine increased
glucose incorporation into glycogen without changing AMKP alpha-1.
Isoleucine but not leucine significantly reduced AMKP alpha-2 activity.
The study suggested that reduced AMPK alpha-2 activity by isoleucine
administration is due to decrease in AMP content and the AMP/ATP ratio
probably because of improvement of ATP availability (Doi et al. 2005).
Isoleucine has been shown to increase the expressions of PPAR-α and
uncoupling protein 2 and 3 (UCP) in high fat diet rats treated for 6
weeks. Hepatic and skeletal muscle triglyceride concentration and
hyperinsulinemia were significantly lower in isoleucine treated rats
which show isoleucine is effective in modifying metabolic syndrome and
preventing obesity (Nishimura et al. 2010).
50
1.13 Summary
It is well documented that both 4-hydroxyisoleucine and isoleucine have
anti-diabetic and anti-dyslipidemic effects. Previous studies have shown
both of these molecules have insulinotropic and insulin-sensitizing
properties which are effective in diabetic and obesity conditions but still
their mechanisms of action remain unclear. Two main differences, based
on well established studies, exist between isoleucine and 4-
hydroxyisoleucine in their mode of action as following:
• Isoleucine elicits its significant hypoglycaemic and anti-
dyslipidemic actions at a higher dose above 100mg/kg (Ikehara et
al. 2008) compared to 4-hydroxyisoleucine which is effective at a
much lower dosage.
• The most important difference between these two molecules is that
isoleucine is hypoglycaemic and anti-dyslipidemic activity is not
dependent on glucose concentration and it can produce its effects
in normal euglycemic conditions whereas the anti-diabetic and
anti-dyslipidemic activity of 4-hydroxyisoleucine is completely
dependent on glucose concentration and it is inactive at normal
glucose concentration less than 6.6 mmol/l. 4-Hydroxyisoleucine's
effects have a direct correlation with glucose concentration in
which higher glucose concentration creates more profound effects
of 4-hydroxyisoleucine (Sauvaire et al. 1998). This feature makes
the 4-hydroxyisoleucine a unique molecule with higher safety and
without any hypoglycaemic side effect in normoglycemic
conditions, unlike most available anti-diabetic medications like
sulfonylureas.
51
All available information about the action of 4-hydroxyisoleucine and
isoleucine are summarised in the following table (Table 1.5). After two
decades from identification of 4-hydroxyisoleucine with unique
properties from fenugreek seed extract still many questions remained
unanswered and mechanisms involve in its glucose dependent anti-
diabetic and hyperlipidemia modifying properties has not been clarified
yet. Understanding mechanisms and modes of 4-hydroxyisoleucine's
actions not only important of its use in management of diabetes but it is a
great help to understand the fundamental metabolic and molecular
pathogenesis and pathophysiology of diabetes which help in designing
new remedies and management approach. All the previous works well
demonstrated the significant hypoglycaemic and lipid lowering activity
of 4-hydroxyisoleucine in both in-vivo and in-vitro conditions in the
presence of insulin which mean 4-hydroxyisoleucine augments
peripheral activity of insulin on glucose uptake. It is claimed that 4-
hydroxyisoleucine anti-diabetic and anti-dyslipidemic activities are due
to its insulinotropic and insulin-sensitizing properties in type 2 diabetes.
There is not sufficient published data about 4-hydroxyisoleucine efficacy
on type 1 diabetes or any high glucose condition without the presence of
insulin. There is also no study on modifying effect of 4-
hydroxyisoleucine on uric acid level which rises in diabetic conditions
specially type 1 diabetes and it is directly linked to diabetic nephropathy
(Rosolowsky et al. 2008).
52
Effect Fenugreek
Seed Extract
4-Hydroxyisoleucine Isoleucine
Blood Glucose level ↓ (Effective in
diabetic, not
normal blood
glucose level)1
↓ (Effective in diabetic,
not normal blood glucose
level) 4
↓(Effective in both
diabetic and
normal blood
glucose level)8
Total Cholesterol ↓1 ↓4 No Data
LDL (Low Density
Lipoprotein)
↓2 ↓4 No Data
HDL (High Density
Lipoprotein )
↑/No change3 ↑/No change5 No Data
Triglyceride ↓ ↓4 ↓9
Uric Acid No Data No Data No Data
Glucose Uptake ↑1 ↑4 ↑8
Glut4 Translocation No Data ↑6 No Data
PI3 Kinase activity No Data ↑7 ↑(Not
Confirmed)10
Glucose Concentration
Dependent
No Data Yes4 No
Insulinotropic No Data Yes4 Yes8
Insuin-sensitizing Action Yes1 Yes4 Yes8
Insulin-independent
hypoglycaemic activity
No Data No Data Yes8
Table 1.5. Summary of findings about fenugreek seed extract, 4-hydroxyisoleucine
and isoleucine based on previous studies covered in the background section.
1- Sharma et al. 1990, Abu saleh et al. 2006, Analava et al. 2006
2- Narendar et al. 2006 3- Gupta et al. 2001
4- Sauvaire et al. 1998, Broca et al. 1999 and 2000
5- Singh et al. 2010 6- Jaiswal et al. 2012 7- Broca et al. 2004
8-Ikehara et al. 2008 9- Nisimura et al. 2010
10- Doi et al. 2003, Zhang et al. 2007
53
1.14 Questions and study design notions
The study in this work was designed to investigate the anti-diabetic
properties of 4-hydroxyisoleucine in-vivo and in-vitro with minimised
insulin effect.
Question 1: Is 4-hydroxyisoleucine capable of decreasing blood glucose
level and modify dyslipidemia, as studied in previous works in type 2
diabetes or insulin-resistant animal models in type 1 diabetes model with
minimal insulin presence? If yes, is it effective in modifying the uric acid
level as well as plasma glucose and lipid levels?
Finding the answer for question 1 is very important which shows that 4-
hydroxyisoleucine is dependent on insulin for its action or not for the
first time in animal models.
Broca showed that infusion of 4-hydroxyisoleucine directly to rat
isolated pancreas islet cells with high concentration glucose stimulates
insulin release (Broca et al. 1999) but there is no other study about the
direct effects of 4-hydroxyisoleucine on pancreatic beta cells. It has been
shown that isoleucine stimulates insulin secretion as well but there is no
direct study on beta cells.
Question 2: Does 4-hydroxyisoleucine increase insulin secretion from
beta cells in the presence of different concentrations of glucose? Does it
increase the consumption of glucose (Uptake) in beta cells? Does
isoleucine increase the insulin secretion and glucose consumption in beta
cells? If yes is is it dependent on glucose concentration? Is isoleucin
action dependent on glucose concentration same as 4-hydroxyisoleucine?
The reason behind the question 2 is based on the fact that insulin
secretion has got direct correlation with glucose concentration and
glucose uptake by beta cells as explained in the early part under insulin
secretion section.
54
There are evidences that 4-hydroxyisoleucine activates insulin signalling
via increasing phosphorylation of PI3Kinase and Akt. It also has been
demonstrated that 4-hydroxyisoleucine modulates glucose consumption
by increasing GLUT4 translocation mediated by PI3Kinase in glucose
disposal cells including adipose tissue and muscle cells (Jaiswal et
al. 2012). The previous study about isoleucine suggested isoleucine
activates PI3Kinase and reduces the activity of AMPK alph-2 due to
decrease of AMP and ATP improvement (Doi et al. 2005).
Question 3: In beta cells GLUT4 is not the major glucose transporter,
and GLUT1 and GLUT2 are mainly responsible for glucose
transportation. If the 4-hydroxyisoleucine and isoleucine increase
glucose consumption in beta cells, it is possibly related to GLUT1 and
GLUT2. Does inhibition of PI3 Kinase in beta cells affect glucose
consumption in beta cells treated with 4-hydroxyisoleucine and
isoleucine?
Based on available knowledge of biochemistry how can we find a
unified explanation for all the effects of 4-hydroxyisoleucine mentioned
in table 5 considering its glucose dependency mode of action? Many
studies found that most of these activities can be explained by the energy
content of the cell. All the signalling pathways within the cells are
handled by a group of enzymes, which are activated and deactivated by
phosphorylation and ATP is the major source of this phosphorylation.
Logically, if the ATP/ADP ratio within the cell is increased by converting
more ADPs to ATPs, it is probable that many of these sensitizing
activities happens. Our aim was to identify any connection between 4-
hydroxyisoleucine and isoleucine and ATP within the cells.
55
Question 4: Do 4-hydroxyisoleucine and isoleucine increase ATP
production and ATP content of beta cells? If yes, can they create the
same effect in other cell types like HepG2 as a cancerous cell with
minimal aerobic respiration? Does PI3Kinase inhibitor affect ATP
production in beta cells treated with 4-hydroxyisoleucine and isoleucine?
As explained before insulin release is completely and directly regulated
by the ATP/ADP ratio. As previous studies claimed on insulinotropic
effect of both 4-hydroxyisoleucine and isoleucine, this effect may be
connected to the ATP level within the pancreatic beta cells. This was the
concept for selecting beta cell as a model to address question 4.
56
Chapter II
Material and Methods
57
Part I
Animal Trial
58
2.1 Animals
Male Wistar rats were purchased from the Pharmacological Research
Centre of Tehran University of Medical Sciences. Eight week-old rats
weighing between 220 and 250 g were used at the start of each treatment
protocol. Rats were housed in separate cages in an animal room kept at
constant temperature (25 ◦C) with a 12 h light–dark cycle. Standard rat
chow pellet and water were provided ad libitum throughout the
experimental period. The animals were maintained in accordance with
the Animal Ethics Committee of the University of Medical Science,
Qom, Iran.
2.2 Preliminary Study
A preliminary pre-experimental screening, animal test was set up to
examine different streptozotocin (STZ) induction protocols for reaching
the optimized condition before proceeding to main experiment. The aim
of the project was to study the effect of 4-hydroxyisoleucine in an animal
model in which there was minimal insulin secretion. STZ was used to
reduce insulin secretion to make it possible to study the effects of 4-
hydroxyisoleucine on glucose metabolism that are independent of
insulin. STZ can be administered intravenously (iv) or intraperitoneally
(ip) to destroy pancreatic beta cells to create a diabetes type 1 rat model.
Intra-peritoneal administration is less effective than intravenous as inject
directly into the blood stream can produce a more profound effect with
lower dose but it was more suitable as it combines less risk of infection
and other side effects in rats during long term experiments because
direct injecting of STZ to vein via rat tail may increase the risk of
transferring germs directly to the blood stream and also there is no option
59
to control bleeding after the injection. STZ was purchased from Sigma-
Aldrich (London, UK) and prepared according to standard protocols
(Motyl et al. 2009) by mixing STZ with freshly prepared Na-citrate
buffer (1.47 g of Na-Citrate into 50 ml deionized water, adjusted PH to
4.5) immediately before injection. Eight rats with weight range of 190-
230 g were divided into four equal groups with two rats in each group.
STZ was injected with doses of single 60mg/kg, single 150 mg/kg and
60 mg/kg for five consecutive days. One group was allocated as control
without STZ injection as vehicle control (injecting Na-citrate buffer
without STZ). Glucose levels were measured using a micro-drop strip
glucometer (Accu-check,Germany) before the injection of STZ and five
days after injection to monitor glucose level. Glucometer was calibrated
by glucose solution prior to test to make sure accurate reading of blood
glucose. The blood glucose levels of all rats were in the range of 68 to 84
mg/dl as an indication of normoglycemic status as explained in chapter
3. Two weeks after receiving the injection rats were killed by volatile
inhalational anaesthetics (Ether) using gas scavenging apparatus for
rodent and blood collected directly from the left ventricle of heart by
blood sampling vacutainer for glucose and insulin measurement. The
blood glucose level increased in all groups apart from the control group.
The blood insulin level was measured using an ELISA insulin kit (DRG
International, NJ, USA) to measure the amount of insulin five days after
the injection as described below.
2.3 Main Experiment
Rats were divided into three groups each of six, comprising normal
controls (NC),diabetic controls (D) and diabetic rats treated with 4HO-
Ile (D4H). Rats were rendered type 1 diabetic by intraperitoneal injection
60
of streptozotocin (60 mg/kg) dissolved in 0.1 M citrate buffer (pH 4.5)
for five days consecutively (Motyl et al. 2009). After one week blood
glucose concentration was measured with a Glucometer on a drop of
blood from the tail. Rats were considered to be diabetic if blood glucose
levels were greater than 300 mg/dl. The treatment group of diabetic rats
were intubated daily with a solution of 4-hydroxyisoleucine in saline
(Extracted from fenugreek seeds with 98% HPLC purity). 4-
Hydroxyisoleucine was a preparation extracted from fenugreek seeds
with 98% HPLC purity (Haeri et al. 2009), and was given at a dose
equivalent to 50 mg/kg/day for four weeks. Normal control rats and
diabetic control rats were intubated with an equivalent volume of saline
alone.
2.4 Insulin Assay
The Blood samples immediately centrifuged at 1000 × g for 15 min.
Plasma was removed and stored at −20 ◦C and insulin level was
measured in each sample by DRG Insulin ultra sensitve ELISA Kit
(DRG International, NJ, USA) which is a solid phase enzyme-linked
immunosorbent assay (ELISA) based on the sandwich principle with
measurement range of 0.02 to 5.5 μg/l . The microtiter wells of kit are
coated with a monoclonal antibody directed towards a unique antigenic
site on the Insulin molecule.
25 μL of each standard, control and samples were dispensed with new
disposable tips into appropriate wells. 25 μL enzyme conjugate was
added into each well and thoroughly mix for 10 second following 30
minutes incubation at room temperature. 50 μL of enzyme complex were
added to each well after 3 times wash with a diluted wash solution (400
μL per well) and incubated at room temperature for 30 minutes. 50 μL of
61
substrate solution were added to each well after 3 times wash and
incubated at room temperature for 15 minutes. Enzymatic reaction was
stopped by adding 50 μL of stop solution to each well and absorbance
(OD) of each well was measured at 450 ± 10 nm with a microtiter plate
reader. A standard curve was constructed by plotting the mean
absorbance obtained from each standard against its concentration with
absorbance value on the vertical (Y) axis and concentration on the
horizontal (X) axis. The concentration of the samples was read directly
from this standard curve.
2.5 Serum lipid profile, glucose and uric acid
At the end of the experiment rats were killed by volatile inhalational
anaesthetics (Ether) using gas scavenging apparatus for rodent and blood
samples were collected by cardiac puncture from the left ventricle into
heparinised tubes and immediately centrifuged at 1000 g for 15 min.
Plasma was removed and stored at −20 °C. Fasting plasma glucose,
triglyceride, cholesterol and LDL concentration were measured on a
fully automated autoanalyser (Biosystem, Spain) by the university of
Qom's medical school reference laboratory.
Plasma uric acid was measured using a timed endpoint method on a fully
automated Beckman Coulter Synchron LX20 clinical system which is an
automated computer-driven, general chemistry analyser designed for the
in vitro determination of a variety of general chemistries, therapeutic
drugs, and other chemistries (Rosolowsky et al. 2008). The tests were
done and verified by university of Qom's medical school reference
laboratory.
62
2.6 Statistical analysis
Data were analysed using SPAW version 18.0 and are expressed as mean
± SD. ANOVA with Tukey and Bonferroni post hoc tests were used to
determine significance of differences between groups. Preventing the
type I error (considering significant a difference that actually isn't
significant) was the main reason we chose to use both Tukey and
Bonferroni post hoc approaches. We tested all the results with Bonferroni
and repeated the analysis with Tukey test to make sure that we make a
correct translation of the results by eliminating type 1 error. P < 0.05 was
considered to be statistically significant.
All the in-vitro results were analysed using only Bonferroni post hoc test.
63
Part II
Laboratory In-vitro Studies
64
2.7 Cell line and cell culture
BRIN-BD11 is a hybrid pancreatic islet cell line formed by the
electrofusion of a primary culture of NEDH rat pancreatic islets with
the rat insulinoma cells (RINm5F cell line). BRIN-BD11 is a stable,
glucose-responsive insulin secreting beta cell line expressing insulin,
glucokinase, Islet Amyloid Polypeptide (IAPP or Amylin) as detected
by immunocytochemistry, and also expresses the GLUT2 glucose
transporter (McClenaghan et al. 1996). BRIN-BD11 was purchased
from the HPA (Health Protection Agency, United Kingdom). 4 x 104
Cells were cultured in RPMI1640 with 2 mM glutamine and 10%
FCS containing 11 mM glucose and incubated in 5% CO2 at 37°C.
The cell population doubled approximately within 24 hours and
reached more than 80% confluency within 48 hours in a 75 cm2 flask.
Cells were seeded for experiments in 24 well plates at 300,000 cells in
each well in 2 ml normal RPMI-1640 with 11 mM glucose for 24
hours (more than 80% confluent). The medium was replaced by
RPMI-1640 with 2 mM glutamine and 10% FCS containing 22 mM
glucose and loaded with treatment compounds at different
concentrations, incubated for 24 hours with 5% CO2 at 37°C. All the
inhibitors and activators used in the current study were loaded at the
same time as isoleucine and 4-hydroxyisoleucine added to culture
media. The glucose concentration of medium with 22 mM glucose
was measured each time before loading using an optimized
glucometer as described below in order to create a baseline for the
glucose concentration at zero time.
65
2.8 Glucose measurement
Glucose levels in the cell culture medium were measured using code-
free glucooxygenase based strip technology. The glucometer
measurement accuracy was evaluated by 50 independent
measurements of glucose in RPMI-1640 containing 11 and 22 mM
glucose separately. The uncertainty intervals and relative deviation
from true value were calculated by following formulation:
Uncertainty Intervals = (highest reading value – Mean)+(Mean- lowest reading value)
Relative Deviation = [(Mean – True Value)/True Value] x 100
The test showed that the machine reading achieved uncertainty
intervals of 0.5 and 0.3 mM and relative deviation of 8.34% and
4.29% in 11 and 22 mM RPMI-1640 respectively. The consistency
and accuracy of reading were important as we used the technique to
compare glucose changes in the medium. The glucose level of each
sample of cell culture medium was measured three times using the
same batch of strips and corrected using relative deviation.
2.9 ATP assay
Cell ATP content was measured by a colorimetric assay from Abcam
(Cambridge, UK) using the manufacturer’s standard protocol as
follows:
300,000 cells were lysed using 100 µl ATP assay buffer provided in
the kit and centrifuged at 15,000 x g and at 4ºC for 2 minutes to pellet
insoluble materials. 50 µl of supernatant was added to each well of a
96-well plate, and the final volume adjusted to 100 µl by adding 50 µl
66
ATP assay buffer. 50 µl of ATP reaction mix containing 44 µl ATP
assay buffer, 2 µl ATP probe, 2 µl ATP converter and 2 µl developer
mix which prepared based on manufacturer protocol provided in the
kit, was added to each well and incubated for 30 minutes at room
temperature. A standard curve was prepared using the standards
provided standards in the kit with the range of 0 to 10 nmol.
Absorption was measured at 570 nm using BMG Labtech FLUOstar
reader.
2.10 Insulin measurement
Insulin in the medium was measured using an HTRF (Homogeneous
Time-Resolved Fluorescence) insulin assay kit from Cisbio
Bioassays, (France) (Youl et al. 2010). HTRF is newly developed
generic assay technology to measure analytes in a homogenous format
by combining fluorescence resonance energy transfer technology
(FRET) with time-resolved measurement (TR). In TR-FRET (HTRF)
assays, a signal is generated through fluorescent resonance energy
transfer between a donor and an acceptor molecule when in close
proximity to each other which significantly reduces the buffer and
media interference using dual-wavelength detection. The final signal
is proportional to the extent of product formation which makes the
HTRF assay sensitive and robust for many antibody-based assays
including the insulin.
The HTRF® insulin assay is a sandwich immunoassay involving two
antibodies, one labeled with Europium Cryptate (Eu-K) and the other
with cross-linked allophycocyanin (XL665). As the two antibodies
bind to insulin molecules, fluorescence resonance energy transfer
(FRET) occurs between the Eu-K and XL665 tracers. This HTRF®
67
signal is thus proportional to the insulin concentration. The
fluorescence emitted from the well is recorded at both 665nm (XL665
emission wavelength) and 620nm (Eu-K emission wavelength) in a
time-resolved manner to differentiate signal from the medium
background and the free acceptor XL665 from the HTRF signal, using
a BMG Labtech FLUOstar plate reader using the following settings:
excitation filter: 337 nm, emission filters: 620 nm and 665 nm;
integration delay (lag time): 60 μs; Integration time:400 μs; number of
flashes: 200; gain: 2300 for 620 and 665 nm. The fluorescence ratio,
expressed as F665 / F620 x10,000, was calculated for each well in order
to minimize medium interferences. A standard curve with a range
from 0 to 12.5 ng/ml was prepared using standard insulin supplied by
the manufacturer of the kit.
50 µl of each sample (Culture medium), 25 µl Ab-XL665 (supplied in
the kit) and 25 μL anti-insulin Ab-XL665 respectively, were added to
each well of 96 well-plate and covered with a plate sealer and
incubated for 2 hours at room temperature then the result read by
BMG Labtech FLUOstar plate reader.
2.11 Compounds
4-Hydroxyisoleucine: Synthetic 4-hydroxyisoleucine was purchased
from TRC, (Noth York, Canada) and had 98% purity as confirmed by
NMR spectroscopic and mass spectrometric analysis, according to the
manufacturer’s data sheet. It contained (2S,3R,4S) 4-
hydroxyisoleucine as the major isomer ( > 94%) and (2R,3R,4S) 4-
hydroxyisoleucine as the minor isomer (< 4%). 100 mM stock
solution was prepared in RPMI-1640 plain medium, filter sterilized
and stored at 4 °C.
68
Isoleucine: Isoleucine was purchased from Sigma-Aldrich (Poole,
UK) and had a purity of 99% (HPLC grade). 100 mM stock solution
in RPMI-1640 plain medium filter sterilized and stored at 4 °C.
Other compounds: the following table contains the list of specialist
compounds used in experiments.
Name Molecular
Weight
Effect Stock solution
Cycloheximide
(Abcam, UK)
281.35 Protein synthesis inhibitor 16mM (10X)
Kaempferol
(Abcam, UK)
286.24 Inhibition of topoisomerase-1, MAO,
COX, estrogenic effects and activation
of the mitochondrial Ca2+ uniporter.
20mM
KU 0063794
(Sigma-
Aldrich, UK)
465.5 Selective inhibitor of mammalian target
of rapamycin (mTOR) (IC50 ~10 nM
for mTORC1 and mTORC2). Displays
no activity at PI 3-kinase or 76 other
kinases tested. Inhibits activation and
hydrophobic motif phosphorylation of
Akt, S6K and SGK, but not RSK.
Suppresses cell growth and induces G1
cell cycle arrest in vitro. (10nM)
10mM
Ruthenium Red
(Abcam, UK)
786.35 Inhibitor of calcium signalling with
multiple actions. Inhibits the
mitochondrial Ca2+ uniporter, Ca2+
dependent ATPase, troponin C and
calmodulin. Attenuates capsaicin-
induced cation channel opening and
inhibits Ca2+ release from
ryanodinesensitive intracellular Ca2+
stores. Blocks large-conductance Ca2+
activated potassium channels.
10 mM(10X)
69
STF-31
(Merck)
423.5 A cell-permeable sulfonamide that
selectively inhibits the growth of VHL-
deficient renal cell carcinomas (RCCs)
dose-dependently (0-5 μM) by directly
targeting glucose transporter 1
(GLUT1), which is up-regulated by
HIF (hypoxia-inducible factor)
transcription factor in VHL-deficient
cells. It elicits decreased oxidative
phosphorylation associated with
aerobic glycolysis and leads to
necrosis, which is consistent with the
effect induced by GLUT1 RNA
interference. It does not bind to other
glucose transporters, and does not
inhibit a broad range of 50 tested
kinases.
10mM
Wortmannin
(Sigma-
Aldrich, UK)
428.43 PI3 Kinase inhibitor 10mM
UK-5099
(Sigma-
Aldrich, UK)
288.3 Inhibitor of plasma membrane
monocarboxylate transporters (MCTs)
and the mitochondrial pyruvate carrier
(MPC).
20mM
70
2.12 Cell metabolism and mitochondrial respiration analysis
The Seahorse Bioscience XF24 instrument was developed and designed
to measure the rate of change of dissolved oxygen and pH in the media
immediately surrounding living attached cells cultured in a microplate in
real time mode. Changes in the extracellular media are caused by the
consumption or production of oxygen and protons by the cells.
Therefore, a sensitive measurement of the media flux can be used to
determine rates of cellular metabolism with great precision and in a
totally non-invasive, label-free manner. Accurate and repeatable
measurements of consumption and production of oxygen and proton in
as little as five minutes are the unique feature of the XF technology. This
is accomplished by isolating an extremely small volume (less than 10 μl)
of media above the cell monolayer. Cellular metabolism causes rapid,
easily measured changes to the “microenvironment” in this small
volume. Typically, a measurement cycle is performed for 2-5 minutes.
During this time, the media are gently mixed and is followed by a short
temperature recovery period. The analyte levels are then measured until
the oxygen concentration drops approximately 20-30% and media pH
declines approximately 0.1-0.2 pH units. The measurement is performed
using 24 optical fluorescent biosensors embedded in a sterile disposable
cartridge that is placed into the Seahorse 24 well tissue culture
microplate. The XF analyser measures oxygen consumption rate (OCR)
as an indicator of mitochondrial respiration and extracellular
acidification rate (ECAR) as the result of glycolysis at intervals of
approximately 2-5 minutes. Baseline metabolic rates typically measure
3-4 times, and are reported in pmol/min for OCR PPR (Protons
Production Rate) and in mpH/min for ECAR. The compound is then
added to the media and mixed for 5 minutes, and then the post-treatment
71
OCR and ECAR measurements are made and repeated. As cells shift
metabolic pathways, the relationship between OCR and ECAR changes.
Cellular oxygen consumption (respiration) and proton excretion
(glycolysis) causes rapid, easily measurable changes to the
concentrations of dissolved oxygen and free protons in this "transient
microchamber" which are measured every few seconds by solid state
sensor probes residing 200 microns above the cell monolayer. Seahorse
analyser continues to measure the concentrations until the rate of change
is linear and then calculates the slope to determine OCR and ECAR,
respectively (Seahorse Bioacience; http://www.seahorsebio.com).
2.13 Optimisation of cell number for Seahorse XF-24
In this experiment, BRIN-BD 11 cells were seeded at four different
densities, 20000, 30000, 40000 and 50000 per well with five
replicates to indicate the best density of cells for seeding to achieve
optimum responses in Seahorse cell culture plates. The basal oxygen
consumption (OCR) rates were measured to the establish baseline rate
in different densities of cells in order to identify the optimum cell
density for experiment repetition.
Cells were dispensed into each well in 100µL RPMI-1640 with 11
mM glucose and left to attach; the plate was left out in the vertical
laminar flow hood (VLFH) for about 30 minutes before returning
them to the incubator as this helped to evenly disperse cells across the
well. All the wells were topped up with medium to 300µL and placed
into CO2 incubator as normal. The growth medium was removed from
each well and the cells were washed with 1ml of pre-warmed RPMI-
1640 without bicarbonate and with 1% FBS because bicarbonate and
72
FBS affect the buffer capacity of the medium and interfere with
protons measurement of the Seahorse XF-24 sensors. Each well was
loaded with 625 μl of RPMI-1640 without bicarbonate and with 1%
FBS medium and incubated in a 37˚C incubator without CO2 for 60
minutes to allow cells to pre-equilibrate with the assay medium.
BRIN-BD11 cells were seeded onto 20 wells of a 24-well Seahorse
special microplate and 4 wells were left and filled with RPMI-1640
only for calibration purposes, as instructed by the manufacturer’s
standard protocol, and shown in the following layout (Fig 2.1). Sensor
Cartridge (Proper orientation: notch at lower left) was loaded to the
machine and pre-warmed RPMI-1640 running medium (without
bicarbonate and with1% FBS) plus 0.3% DMSO was loaded to
control wells (A1, B4, C3 and D6). The Seahorse XF-24 command
was adjusted to 3x loop start and 3 minute measurement at each loop.
Figure 2.1. The layout of negative controls in the Seahorse microplateplate. The
green wells were filled with RPMI-1640 medium without cells for the purpose of
machine calibration during test runs.
73
2.14 Results of cell number optimization
The results of two preliminary experiments to verify the best density
of BRIN-BD 11 cells for use in Seahorse XF-24 microplates showed
that the density of 40000 cells per well achieved the best oxygen
consumption rate (OCR) measurements compared with other cell
densities within the machine reading range (Fig 2.2). The density of
50000 cells produced out of range results. As a conclusion, the density
of 40000 BRIN-BD11 cells is the maximum cells which can be
loaded to create enough cells for a robust mitochondrial respiration
measurement with Seahorse XF-24. To eliminate probable bias, five
replicates for each density were measured simultaneously.
74
a)
b) Red: 20K Blue: 30K Green: 40K (Green in the bottom is baseline without cells)
Figure 2.2. Five replicates of 20000, 30000, 40000 and 50000 BRIB-BD 11 cells
were seeded respectively from column A to D of the microplate. A1, B4, C3 and D6
were filled by RPMI-1640 without cell for calibration during the test (a). The
measurements of oxygen consumption rate (OCR) in 50 minutes shows the density
of 40000 BRIN-BD11 cell creates more robust reading compared to others and
density of 50000 cells creates out of range result (b).
75
Chapter III
Animal Trial
All the facilities for carrying this animal study were provided by Qommedical university, Iran and Dr Mohammadreza Haeri. He also helped
me in all the steps of handling animals, collecting samples and analysingdata. Dr Kenneth White also provided me with immense help instatistically analysing data and writing the paper which has been
published in Phytomedicine journal (Appendix I)
76
3.1 Type 1 diabetes animal model
The main characteristic of type 1 diabetes is a lack of insulin production
due to the destruction of the pancreatic beta cells, which can be achieved
in animal via chemical destruction of beta cells or breeding a genetically
modified rodent who develop pancreatic beta cell destruction due to
spontaneous autoimmune response. Steptozotocine (STZ) and alloxan
are two main compounds which use for chemically induced type 1
diabetes. They compete with glucose to enter pancreatic beta cells due to
their structural similarity with glucose. Chemically induced diabetes is
an easy and cheap technique to create a type 1 diabetes model for testing
drugs or therapies where the main mechanism of action is lowering
blood glucose in a non beta cell dependent or insulin independent
manner (Sheshala et al., 2009). Both STZ and alloxan can be toxic at
other organs of the body and this should be considered when drugs are
being tested in these models. STZ tolerates better than alloxan and has a
wider diabetogenic ratherthan alloxan with a narrow diabetogenic dose.
Alloxan light overdosing can cause general toxicity, especially to the
kidney (Szkudelski,2001).
Streptozotocin (STZ) is particularly toxic to pancreatic beta cells and has
been used for several decades to induce the diabetic state in rodents
(Davidson et al. 1977, Rees et al. 2005). STZ is a glucosamine
nitrosourea compound similar to glucose, that is recognised and
absorbed by GLUT2, but not other glucose transporters. STZ induces
DNA damage by activation of poly ADP-ribosylation within the beta
cells (Szkudelski. 2001), and is particularly toxic to pancreatic beta cells
because they express high level of GLUT2 (Wang et al.1998).
77
Figure 3.1. Streptozotocin chemical structure. (Adapted from Sigma Aldrich,
http://www.sigmaaldrich.com)
The type 1 diabetes rat model can be produced using single high dose STZ
and multiple injection of STZ over 5 days. Single high dose STZ
administration could create a simple hyperglycaemia model, but it is not
stable for long term model due to spontaneous regeneration of endogenous
beta cells (King. 2012). Multiple injection of STZ could create more stable
damage to pancreatic beta cell which could not be regenerated after a few
weeks (King. 2012). Chemically induced animal is not a appropriate
model for studying the pathogenesis of type 1 diabetes. The spontaneous
models of autoimmunity is more close to human disease for such study
(Reddy et al. 1995). We adapted the STZ technique to create a suitable
model for our study as our aim is to investigate the effect of 4-
hydroxyisoleucine on blood glucose level in an animal model close to type
1 diabetes not studying the pathogenesis of the disease.
We were looking to create a type 1 diabetes rat model with minimum
insulin secretion in which the animal can survive throughout the
experimental period without the need to administer insulin. This would
allow us to assess the effect of 4-hydroxyisoleucine on condition with a
significant reduction in insulin production ability and the amount of
insulin in circulation.
We compared three protocols of STZ administration, using intra-
peritoneal (IP) injection to achieve a stable model with high blood
78
glucose level and significantly reduced insulin production capability. A
single IP injection of STZ at doses of 60 mg/kg or 150 mg/kg were
compared with the procedure of daily single IP injections of STZ at 60
mg/kg for five consecutive days (Motyl et al. 2009). We ran a
preliminary test to compare these different protocols to achieve the best
possible selection of a suitable rat model for the study. Eight rats with
the weight of 190-230 g were selected and divided into 4 equal groups
with two rats in each group (Table 3.1). The glucose level was
measured using a micro-drop strip glucometer before the injection of
STZ, at day 5 after injection. The rats were killed two weeks after
injection and blood collected directly from the heart for glucose and
insulin measurement.
Group A Single dose STZ 60 mg/kg IP injection
Group B Single dose STZ 150 mg/kg IP injection
Group C Multiple dose STZ 60mg/dl consecutive 5 days IP Injection
Group D Normal Control without STZ
Table 3.1. Different protocols of intraperitoneal injection of STZ.
Single doses of STZ induced increases in glucose levels, but not to
levels greater than 300 mg/dl, the threshold for a diabetic phenotype
(Table 3.2). Repeated doses of STZ induced glucose levels of 520 ± 13
mg/dl compared with 79 ± 3 mg/dl for controls (n = 2), and insulin
levels were below the limit of detection (0.2 µg/l) using automated
clinical reader compared with an average of 1.79 µg/l in controls. We did
not use ultra sensitive ELISA kit for preliminary study for its high cost, but
the method of detection was accurate enough for our purpose. We used
different insulin measurement using ultra sensitive rat insulin kit with
detection range of 0.02 µg/l to 5.5 µg/l. On this basis the repeated doses of
79
STZ could be used to induce a state in rats as close as possible to type 1
diabetic. Multiple STZ injection created a desired model with
hyperglycaemia and minimized insulin production, which suggest a
massive destruction of pancreatic beta cells. Using such model could make
it possible the activity of 4-hydroxyisoleucine in different angle.
Group Blood Glucose
mg/dl
Blood Glucose
mg/dl
Blood Glucose
mg/dl
Insulin
µg/l
A1
A2
82
77
SD ± 3.54
118
109
SD ± 6.36
127
123
SD ± 2.83
1.19
0.94
SD ± 0.77
B1
B2
68
79
SD ± 7.78
201
177
SD ± 16.97
247
262
SD ± 10.61
0.59
0.51
SD ± 0.06
C1
C2
71
84
SD ± 9.19
497
441
SD ± 39.6
530
511
SD ± 13.4
lower than
measurement level
D1
D2
71
76
SD ± 3.54
80
73
SD ± 4.95
77
81
SD ± 2.83
1.81
1.76
SD ± 0.04
Table 3.2. Comparison of blood glucose and insulin in rats treated with different STZ
injection protocols. Two rats were allocated in each group as explained in Table 1.
Blood glucose levels before injection, in day 5 and two weeks after injection was
shown in columns 2, 3 and 4 respectively. Group C shows highest sustained blood
glucose level with no trace of insulin within 2 weeks of 60mg/kg STZ injection for
five consecutive days. Rats in group C survived with high blood glucose level for
two weeks and it demonstrates a suitable diabetic rat model close to diabetes type 1
for our experiment .
80
3.2 Animal study results and discussion
Rats were divided into three treatment groups, each with six animals.
Two groups were treated with a single dose of 60 mg/kg STZ for
five consecutive days and the third group received a vehicle control.
Animals treated with STZ were left for seven days to develop diabetes,
as confirmed by measurement of blood glucose (glucose level of tail
blood over 300 mg/dl) before starting treatment with 4-
hydroxyisoleucine. The diabetic treatment group was intubated with 50
mg/kg/day of 4-hydroxyisoleucine, and the diabetic control group with
saline vehicle, continuously for four weeks. One week after STZ
administration rats had a markedly elevated plasma glucose compared
with controls, and this was sustained for a further four weeks (Fig 3.1,
groups D and D4H). Fasting blood glucose, triglyceride, cholesterol,
LDL, HDL and uric acid were measured using methods described in
chapter II and data analysed statistically with the ANOVA Bonferroni
and Tukey tests to avoid type 1 error. The correlation with P value less
than 0.05 in both tests was accepted as significant. Treatment of
diabetic rats with 4-hydroxyisoleucine for four weeks induced a
reduction in blood glucose from 500[±SD] mg/dl to 330[±SD] mg/dl
which is statistically significant (P<0.05). The blood glucose level in
both control and diabetic control groups remained steady during the
experimental period without significant changes, comparing blood
glucose levels at the beginning and the end of the experiment in each
group (Fig 3.1). It shows that damaged pancreatic beta cells were not
restored naturally in the STZ induced diabetic group during the
treatment period of four weeks and the diabetic status was constant.
The fact that blood glucose levels remained steady over the four week
period of the experiment in the non-diabetic group, after being housed
81
in the same conditions as the other groups, suggests that the prevailing
environmental and dietary conditions did not influence blood
glucose levels. Generally the diabetic animals treated with 4-
hydroxyisoleucine had an improved appearance and it was
noticeable that the heavy ocular vascularization induced by STZ was
being reversed as the treatment with 4-hydroxyisoleucine progressed
(Haeri et al. unpublished data).
Insulin was measured by ultrasensitive ELISA kit with the detection
limit of 0.02 µg/l to 5.5 µg/l. All the samples measurement were
within the detection range. Insulin levels in the diabetic groups
were significantly lower than normal controls with levels decreased
by 60 % from 0.7 µg/l to less than 0.3 µg/l. There is not a
significant difference in insulin levels between the diabetic control
and diabetic group treated with 4-hydroxyisoleucine, after four
weeks (Fig 3.2).
These data suggest that restoration of pancreatic beta cells does not
play a role in improving diabetic conditions in the 4-
hydroxyisoleucine treatment group.
82
Figure 3.1. Fasting plasma glucose at the beginning and end of the 4-
hydroxyisoleucine treatment. Mean and SD plasma glucose of five or six rats per
group. Control rats (C). Diabetic (D) and treatment (D4H) groups were made diabetic
by repeated doses of STZ. Treatment with 4-hydroxyisoleucine started one week
after the STZ treatment and continued for four weeks. Glucose was measured at the
start of the 4-hydroxyisoleucine treatment (White Columns) and at the end of four
weeks treatment (Grey Columns). 4HO-Ile induced a significant (*p < 0.05) decrease
in plasma glucose within the treatment group after four weeks.
83
Figure 3.2. Plasma insulin after treatment with 4-hydroxyisoleucine. Plasma insulin
was measured by ultrasensitive insulin ELISA kit with the detection limit of 0.02
µg/l to 5.5 µg/l. Data are mean + SD of five or six rats per group at the end of the
treatment period. Insulin levels were significantly lower in diabetic groups [D, D4H]
compared with control [C] (*p < 0.05).
The diabetic rats had a markedly higher intake of food and water
compared with controls (Fig 3.3), but the hyperphagia did not cause any
increase in body weight in the diabetic rats compared with controls.
Body weights (mean ± SD) at the end of the study were 289 ± 31 g (C),
269 ± 15 g (D) and 267 ± 5 (D4H). There was no significant difference
between groups.
84
Figure 3.3. Water and food intake over treatment period. Intake of water (a) and food
(b) over the four week period of 4-hydroxyisoleucine treatment. Data are mean + SD
daily intake per animal over weekly periods for control (C), diabetic (D) and diabetic
rats treated with 4-hydroxyisoleucine (D4H). Data are for the four consecutive weeks
of the period of treatment with 4HO-Ile: week 1 ( ), 2 ( ), 3 ( ), 4 ( ). Significant
differences between means of group D compared with groups C or D4H, for a given
week, are indicated where p < 0.05 (*) or p < 0.01 (**).
85
The lipid profile of the diabetic rats was also consistent with a diabetic
phenotype, with significantly elevated triglyceride, cholesterol, HDL
(High Density Lipoprotein) and LDL (Low Density Lipoprotein)
compared with the control group (Fig 3.4). The changes in glucose and
lipids coupled with the marked decrease in insulin indicate that a type 1
diabetic phenotype is induced by the repeated intraperitoneal doses of
STZ. The levels of triglyceride, LDL and HDL in the treated diabetic
animals were not significantly different to those of control, non-diabetic
rats and total cholesterol was reduced to near control levels (Fig 3.4b)
More strikingly the 4-hydroxyisoleucine treatment also resulted in
significant decreases in all lipid markers compared with untreated
diabetic rats.
The levels of triglyceride, LDL and HDL in the treated diabetic animals
were not significantly different to those of control, non-diabetic rats and
total cholesterol was reduced to near control levels indicating that
treatment with 4-hydroxyisoleucine had restored the diabetic lipid profile
to an almost normal one. In type 1 diabetes levels of HDL typically
decrease but in our model the STZ-treated animals had elevated HDL.
The reason for this is unclear, but similar behaviour in STZ-rats have
been found by others (Islam. 2011). In our study the treatment with 4-
hydroxyisoleucine could reverse the changes in HDL induced by STZ
and restore levels close to that of control non-diabetic animals (Fig 3.4).
86
Figure 3.4. Serum lipid profile of rats after four weeks treatment with 4-
hydroxyisoleucine. Triglyceride (a), cholesterol (b), LDL (c) and HDL (d) measured
at the end of the treatment with 4-hydroxyisoleucine are shown as the mean + SD of
five or six rats per group. Levels were significantly lower in control (C) and treated
diabetic (D4H) groups compared with untreated diabetic animals (D) (*p < 0.05, **p
< 0.01).
Uric acid levels in the STZ-induced diabetic rat group were elevated
significantly, about 40% compared to the non-diabetic group, and this
increased hyperuricemia of the diabetic rats was restored to levels
found in normal rat controls after treatment with 4-hydroxyisoleucine
(Fig 3.5). There is a relationship and biochemical interaction between
high serum glucose and high serum uric acid levels as uric acid is
87
increased during hyperglycaemia (Cook et al. 1986). The relationship
between serum uric acid and metabolic diseases has been explored in a
number of studies. There is a positive relationship between levels of
blood insulin and blood uric acid level and uric acid concentration is
closely related to pancreatic beta cell activity (Robles-Cervantes et al.
2011, Sinagra et al. 1996). Hyperuricemia is directly linked to damage
to the kidney, which is one of the major causes of morbidity and
mortality in diabetes (Rosolowsky et al. 2008). There is increasing
evidence from clinical data that high levels of uric acid are an
independent risk factor for kidney disease (Miao et al. 2011). It also has
been demonstrated that high blood uric acid level is directly associated
with macro and microangiopathies and also independently associated
with coronary heart disease and renal dysfunction in diabetic patients
(Ito et al. 2011). It is notable in this study that the reduction in
hyperuricaemia caused by treatment with 4-hydroxyisoleucine is more
pronounced than the reduction in hyperglycaemia, in that uric acid levels
return to those of normal controls (Fig 3.5 compares groups C and
D4H) whereas levels of blood glucose remain three times higher than
normal controls (Fig 3.1 compare groups C and D4H) and insulin
levels remained unchanged (Fig 3.2 compare groups D and D4H). It
remains to be seen whether or not the effects of 4-hydroxyisoleucine on
serum uric acid are mediated through improved, but not fully restored
glucose levels, or by another mechanism without increasing insulin level.
88
Figure 3.5. Serum uric acid after four weeks treatment with 4-hydroxyisoleucine.
Data are mean + SD serum uric acid in rats at the end of the period of treatment with
4-hydroxyisoleucine. Levels were significantly lower in control (C) and treated
diabetic (D4H) groups compared with untreated diabetic animals (D) (*p < 0.05).
The effective correction of the diabetes induced dyslipidemia by 4-
hydroxyisoleucine is similar to results of the 4-hydroxyisoleucine
treatment of rats or mice with a type 2 diabetes phenotype, in which 4-
hydroxyisoleucine induced improvements in both lipid profile and
hyperglycaemia (Haeri et al. 2009; Singh et al. 2010). anti-
dyslipidemic activity of 4-hydroxyisoleucine has also been demonstrated
in a hamster model of dyslipidemia (Narender et al. 2006). The findings
presented in this thesis show that 4-hydroxyisoleucine is capable of
modifying diabetes type 1 condition as a new mode of action in addition
to the known beneficial effects of 4-hydroxyisoleucine in type 2 diabetes.
Previous studies of the anti-diabetic properties of 4-hydroxyisoleucine
have focused on diabetes type 2 and animal models of insulin resistance
and have suggested that 4-hydroxyisoleucine produces its anti-diabetic
89
effects via stimulating insulin secretion from pancreatic beta cells
(Insulinotropic) and by an insulin sensitizing mechanism (Sauvaire Y et
al. 1998, Broca et al. 1999, Broca et al. 2000, Jette et al. 2009). In the
work presented in this thesis it is notable that 4-hydroxyisoleucine did
not induce an increase in insulin levels in diabetic rats compared with
untreated diabetic controls (Fig 3.2). Both groups had insulin levels of
about 0.3 g/l, which were approximately 65% lower than the non-
diabetic control group (Fig 3.2).
These data show that 4-hydroxyisoleucine has no insulinotropic activity
in this model of type 1 diabetes, despite the high levels of glucose in the
diabetic rats. Thus, even though insulin levels were low, indicative of a
small amount of pancreatic activity, they were unchanged by treatment
with 4-hydroxyisoleucine.
The improvement by treatment with 4-hydroxyisoleucine on the
metabolic parameters of glucose, lipid profile and uric acid in a model of
type 1 diabetes model with low level of insulin availability and
pancreatic beta cell activity as well as diabetes type 2 and insulin
resistant conditions suggest that 4-hydroxyisoleucine has a systemic
effect on metabolically active tissues, including liver, muscle and
adipose tissue, that is independent of insulin. The data from our study
and from previous literatures cited above suggest that 4-
hydroxyisoleucine could be used as a treatment for both type1 and type
2 diabetes independently or in combination with other treatments.
Analysing the data from the current study show that 4-
hydroxyisoleucine's main mechanism of action is not insulin stimulating
or insulin sensitizing as was determined in previous studies.
90
We assume that 4-hydroxyisoleucine produces its anti-diabetic effects
by facilitating glucose utilization in tissues independent of insulin
considering the fact that 4-hydroxyisoleucine exhibits its activity in a
high glucose concentration without glucose lowering effect in normal
blood glucose level (Sauvaire et al. 1998). 4-hydroxyisoleucine could
be a promising candidate for of diabetes treatment and understanding its
mechanism of action to control diabetes metabolic disturbances also
could shed a light to new treatment approaches and designing new
medications.
91
3.3 Animal study findings at a glance
• 4-hydroxyisoleucine is effective in modifying diabetes type 1
metabolic conditions as well as those of diabetes type 2 and
insulin resistance.
• 4-hydroxyisoleucine produces its anti-diabetic effects independent
of insulin as no increase in insulin was observed through the
duration of the study. It challenges the previously
suggested mechanism of action such as an insulinotropic
activity for 4-hydroxyisoleucine.
• 4-hydroxyisoleucine restores LDL (Low Density Lipoprotein)
and triglyceride close to normal levels in diabetic rats, which
demonstrate its effectiveness in reducing the risk of cardiac and
vascular complications of diabetes.
• The effect of 4-hydroxyisoleucine on uric acid has been studied
for the first time and it was demonstrated that 4-hydroxyisoleucine
can restore diabetic hyperuricemia without increasing insulin
secretion or pancreatic beta cell activity.
92
Chapter IV
In-Vitro Study
BRIN-BD11 Cell
In-vitro studies discussed in this chapter have been carried out with the
help of my colleague, Dr Ravi Velga. He helped me during all the steps
for preparation, cell culture and running tests. I would like to thank him
for all his help. I would like to thank Dr Kenneth White for his
constructive advices throughout this phase of the study.
93
4.1 Introduction
The animal studies described in Chapter III showed that 4-
hydroxyisoleucine decreases plasma glucose, concentration and
modifies other diabetes-related adverse effects such as hyperlipidemia
and hyperuremia, independently of insulin. This mode of action
contrasts with previous suggestions that 4-hydroxyisoleucine’s mode
of action is as an insulinotropic and insulin sensitizing agents (Broca
et al. 2004, Sauvaire et al. 1998, Al-Habori et al. 1998), and raises the
question as to how 4-hydroxyisoleucine decreases blood glucose
levels. Interestingly, isoleucine, which is structurally close to 4-
hydroxyisoleucine, also had an insulin-independent hypoglycaemic
effect on diabetic mice (Ikehara et al. 2008), and it increased glucose
uptake in muscle of fasted rats without changing insulin levels (Doi et
al. 2007). Accordingly isoleucine was included in cell based studies,
described below, which attempted to elucidate the mechanism of
action of 4-hydroxyisoleucine.
In view of the published literature about isoleucine and of the data from
our animal study, we hypothesized that 4-hydroxyisoleucine
increases glucose uptake and utilisation in peripheral tissues, which
leads to its anti-diabetic effects. Evidence to support the hypothesis
that 4-hydroxyisoleucine increases glucose utilization in peripheral
tissues which is not insulin mediated includes the observation that
chronic, but not acute, treatment with 4-hydroxyisoleucine produces
hypoglycaemia (Broca et al. 2004), that the activity of 4-
hydroxyisoleucine is insulin independent (Haeri et al. 2012), that the
anti-diabetic effects of 4-hydroxyisoleucine directly correlate with
glucose concentration (Sauvaire et al. 1998) and that the chemical
structure is similar to isoleucine, which has been shown to increase
94
glucose uptake independent of insulin (Doi et al. 2007). To
investigate the insulin-independent effects of 4-hydroxyisoleucine and
isoleucine on glucose uptake and utilization a cell model was used
which is not insulin-responsive and dependent on the insulin signalling
pathway for glucose uptake. Use of the model provides the opportunity
to study the mechanisms of action of both isoleucine and 4-
hydroxyisoleucine independently.
The model will also shed light on the question whether 4-
hydroxyisoleucine is an insulin sensitizing agent, or whether its
independent effect on increasing basal glucose utilization facilitates
insulin action as well - if any molecule can increase glucose consumption
independently in peripheral tissues, it can increase the insulin sensitivity.
4.1.1 BRIN-BD 11 cell as a model for In-vitro study
Neural cells, kidney tubular cells and pancreatic beta cells all possess an
insulin-independent glucose uptake mechanism. Neural cells possess both
insulin independent and insulin mediated glucose uptake mechanisms
(Schulingkamp et al. 2000). Glucose uptake in pancreatic beta cells is not
mediated by insulin due to their glucose sensory nature and any insulin
mediated glucose uptake mechanism would interrupt the sensory function.
Glucose uptake is independent of insulin in pancreatic beta cells. Insulin has
autocrine activity on beta cells as a modulator, and has different roles
such as enhancing beta cell survival and inducing proliferation,
through mechanisms that are unclear (Emilyn et al. 2010).
In contrast to adipose tissue and muscle cells, which express high levels
of GLUT 4, an insulin regulated glucose transporter, the glucose
transporters mainly expressed in beta cells are GLUT 1 and GLUT 2. In
95
mouse beta cells GLUT 2 is expressed at higher levels than GLUT 1 and
is responsible for glucose-stimulated insulin secretion, while in human
beta cells both GLUT 1 and GLUT 2 are responsible for glucose-
stimulated insulin secretion (Ohtsubo et al. 2011).
Considering the physiological and biological properties of a variety of
cell lines, beta cells make an appropriate model for investigating the
effects of 4-hydroxyisoleucine and isoleucine on glucose uptake by
minimising the interference of insulin. The search for a cell model that has
the normal functionality of beta cells, with a well established record
and the ability to be grown in normal laboratory conditions, guided us
towards the BRIN-BD11 cell line. BRIN-BD11 is a hybrid cell line
formed by the electrofusion of a primary culture of NEDH rat pancreatic
islets with RINm5F (a cell line derived from a NEDH rat insulinoma)
and unlike other tumoral cell lines, it spreads and grows evenly in
culture as monolayers with an epithelioid morphology (McClenaghan et
al. 1996).
BRIN-BD11 cells also have the unique advantages that they retain key
attributes of normal pancreatic beta cells for glucose responsiveness,
insulin synthesis and secretion (McClenaghan et al. 1996). They are also
immortalised and provide stable growth and function up to 50
passages. Other attributes include a high capacity for glucose sensing,
specificity, transport and metabolism, and appropriate responses to
a range of non-glucose nutrients and receptor-mediated modulators of
beta cell function, including amino acids, neurotransmitters and
sulphonylurea drugs. Preliminary studies showed that amino acids
may act on BRIN-BD11 cells by diverse mechanisms, including
metabolism to ATP and uptake of cationic amino acids (McClenaghan et
96
al. 1996). BRIN-BD11 cells are also suitable for nutrient
interaction studies of the regulation of insulin secretion by
sulphonylureas and other compounds.
97
4.2 The effect of 4-hydroxyisoleucine and isoleucine on glucose
consumption in BRIN-BD11 cells
In the first set of experiments using BRIN-BD11 cells, different
concentrations of glucose were used to assess the effects of 4-
hydroxyisoleucine and isoleucine on glucose uptake.
The glucose concentration of the culture medium is one of the
criteria considered to model the diabetic status in order to evaluate the
effect of isoleucine and 4-hydroxyisoleucine on glucose
consumption. Exposure of BRIN-BD11 to 500 µM of isoleucine and 4-
hydroxyisoleucine added to RPMI-1640 medium contains 5, 11 and
22 mM of glucose for 24 hours, showed a very significant increase in
glucose consumption in cells incubated with 4-hydroxyisoleucine with
22 mM glucose RPMI1640 compared with control and other
groups (Fig 4.1). Glucose consumption increased constantly and
significantly in all groups compared to 4-hydroxyisoleucine which
showed activity in 11 and 22 mM glucose containing medium.
The results of the first experiments demonstrated the correlation
between 4-hydroxyisoleucine activity and glucose concentration, in
which 4-hydroxyisoleucine is only active at higher concentrations of
glucose and the activity increases with increase in glucose, and are
consistent with previous observations (Sauvaire et al. 1998). The effect
of isoleucine, to stimulate a relatively constant increase in glucose
uptake in all concentrations of glucose, indicates its activity is
independent of glucose concentration.
98
Figure 4.1. The Effect of Ile and 4HO-Ile on glucose consumption by BRIN-
BD11 cells. Comparing glucose consumption in BRIN-BD 11 cells incubated with
500 µM isoleucine and 4-hydroxyisoleucine for 24 hours in variable concentrations
of 5, 11 and 22 mM of glucose in RPMI-1640 medium. Glucose consumption is
increased very significantly by both 4-hydroxyisoleucine and isoleucine in 22 mM
group (p<0.01) and significantly in 11 mM group (p<0.05). The experiment was
repeated three times with five sets of replicate in each experiment and results analysing
by ANOVA Bonferroni test. Isoleucine shows a consistent increase in glucose
consumption independent of glucose concentration compared to 4-hydroxyisoleucine
which its activity increased correlatively with glucose concentration. Both 4-
hydroxyisoleucine and isoleucine exhibit optimal activity in 22 mM concentration of
glucose.
99
4.3 The effect of 4-hydroxyisoleucine and isoleucine on insulin
secretion from BRIN-BD11 cells
The experiment was set up to assess the effect of 4-
hydroxyisoleucine and isoleucine on insulin secretion in BRIN-
BD11 cells. The amount of insulin within the medium has been
measured as a reflection of insulin secretory activity of BRIN-BD 11
cells. There was no significant increase observed in insulin secretion
between 4-hydroxyisoleucine and isoleucine compared to control in all
groups (Fig 4.2). The increase in insulin secretion was consistent with
the increase in glucose concentration, as expected, which
confirms the normal glucose responsiveness of the insulin
secretion activity of BRIN-BD 11 cells and hence the reliability of
using BRIN-BD 11 cells as a model for normal pancreatic beta cells.
Figure 4.2. The Effect of isoleucine and 4-hydroxyisoleucien on insulin
secretion by BRIN-BD11 cells. The measurement of insulin in cell culture medium
showed a very significant increase in insulin secretion by increasing glucose
concentration (p<0.01). There is no significant differences in insulin secretion in cells
incubated with 500 µM 4-hydroxyisoleucine and isoleucine for 24 hours. The
experiment was repeated three times with five sets of replicate in each experiment
and data were analysed by ANOVA Bonferroni test.
100
Insulin measurements revealed that both 4-hydroxyisoleucine and
isoleucine did not stimulate insulin secretion, whilst they both
increased glucose uptake (Fig 4.1). This finding confirms the results
from animal studies in which 4-hydroxyisoleucine lowered blood
glucose without increasing plasma insulin. The current results from
BRIN-BD 11 cells and animal studies do not support the findings of
Sauvaire in 1998 and Broca 1999 (Sauvaire et al. 1998, Broca et al.
1999) which suggested that 4-hydroxyisoleucine produces its anti-
diabetic effect by stimulating insulin release from pancreatic beta cells.
Current observations reconfirm an insulin independent mechanism of 4-
hydroxyisoleucine in utilizing glucose consumption. A similar
conclusion was arrived at when it was shown that 4-
hydroxyisoleucine stimulated glucose uptake in skeletal muscle L6
cells in the absence of insulin (Jaiswal et al. 2012).
Significant increase in glucose consumption in beta cell exposed to high
concentration of glucose (22 mM) and long incubation with 4-
hydroxyisoleucine without changing insulin secretion explains an
insulin-independent glucose utilization mechanism involves in 4-
hydroxyisoleucine anti-diabetic effects. Isoleucine follows the pattern of
4-hydroxyisoleucine with a substantial difference in dependency to
glucose concentration. The glucose concentration dependent activity of
4-hydroxyisoleucine makes it an interesting molecule with a unique
mode of action, only active in hyperglycaemic condition and does not
show hypoglycaemic effect in lower glucose concentrations. It is active
when it needs to create an effect.
101
4.4 The effect of 4-hydroxyisoleucine and isoleucine on ATP content
of BRIN-BD11 cells
Incubation of BRIN-BD 11 cells with 4-hydroxyisoleucine and
isoleucine did not increase ATP levels in BRIN-BD 11 cells after 24
hours whilst the glucose uptake was increased. The increase of ATP level
was in direct correlation with the glucose concentration of the culture
medium as expected (Fig 4.3). Doi's study shows that isoleucine did not
change the ATP level in liver cells whilst it increases glucose uptake (Doi
et al. 2007). Current study iterates that isoleucine produces the similar
effect in beta cell as well as observed in liver cell. Exposure of the beta
cell to higher glucose concentration stimulates insulin secretion by
increasing the ATP production within the beta cell and the amount of
insulin secreted from cell has a direct correlation with glucose
concentration. ATP measurement results in BRIN-BD 11 also showed
that insulin secretion increases in higher concentrations of glucose (Fig
4.2) which suggest that BRIN-BD11 resembles the normal beta cell
attributes.
The glucose concentration of 22 mM was adapted as a standard glucose
concentration for all the experiments during the current study. There
were no significant differences observed in glucose consumption in
BRIN-BD 11 in the presence of 22 mM glucose between the variable
range of 4-hydroxyisoleucine and isoleucine concentration from 100
micro molar to 1 mM. It is in line with Sauvaire et al study in 1998
which suggested, 4-hydroxyisoleucine glucose lowering activity is
present in concentration range from 100 micro molar to 1 mM. This
finding also shows that 4-hydroxyisoleucine dose-dependent effect
pattern in lower concentration (lower than 50 micro mole) as observed in
102
Jaiswal et al in 2012, is not seen in dose above 100 micro mole up to
1mM. The concentration of 500 micro molar of both 4-
hydroxyisoleucine and isoleucine was used for all the future experiments
in this study as an average effective dose. The pattern of glucose
consumption and cellular ATP level were consistent with the findings as
shown in figure 1 and 2 in all the run through the study (Fig 4.4).
Figure 4.3. The Effect of isoleucine and 4-hydroxyisoleucine on ATP content
of BRIN-BD11 cells after 24 hours. ATP level in BRIN-BD 11 cell measured after
24 hours with HTRF ( homogeneous time-resolved fluorescence) technique. There is
a very significant increase in the amount of ATP within the cells in medium with11
mM and 22 mM glucose concentration compared to each other and medium with
5mM glucose (p<0.01). There is no significant difference in ATP level of 4-
hydroxyisoleucine and isoleucine groups. The experiment was repeated three times
with five sets of replicate in each experiment and data were analysed by ANOVA test.
103
a) b)
Figure 4.4. Glucose consumptions in the same batch of BRIN-BD 11 cells used
for ATP content measurements. Glucose consumption in BRIN-BD11 cells
increases very significantly in treatment group with 500 µM 4hydroxyisoleucine
(p<0.01) and significantly with 500 µM isoleucine (p<0.05) compared to control
group after 24 hours in the presence of 22 mM glucose (a). There is no significant
changes in ATP level between groups (b).
104
4.5 Cycloheximide (CHX) and protein synthesis role in 4-
hydroxyisoleucine and isoleucine mechanism of actions
A recent published study by Jaiswal et al in 2012 showed that increase in
glucose uptake and GLUT 4 translocation in L6-GLUT4myc skeletal
muscle cells in the presence of 4-hydroxyisoleucine are blocked by
adding 1 microg/ml of cycloheximide (CHX) which is a potent protein
synthesis inhibitor. CHX is a selective inhibitor of eukaryotic protein
synthesis which blocks tRNA binding and release from ribosome (Obrig
et al. 1971). Incubation of BRIN-BD11 cells with 4hydroxyisoleucine
and isoleucine in the presence of 0.8 μM (1 µg/ml) of CHX showed that
CHX significantly inhibits the effect of 4-hydroxyisoleucine on
increasing glucose uptake but not isoleucine (Fig 4.5). ATP measurement
also did not show any changes in the content of ATP within the cells in
front of CHX after 24hours (Fig 4.5).
Isoleucine significantly increases the glucose consumption in the
presence of CHX, suggests a synergistic effect in isoleucine mechanism
and also signifies that isoleucine glucose lowering effect does not
dependent in synthesis of new proteins whilst 4-hydroxyisoleucine
mechanism is noticeably dependent on protein synthesis in beta cells and
skeletal muscles which previously observed by Jaiswal (Jaiswal et
al. 2012). Observing the same result in different cells and different dose
extends the conclusion that 4-hydroxyisoleucine hypoglycaemic effect
and in some extent anti-diabetic properties directly or indirectly are
mediated by a protein or proteins which need to be synthesised. It can be
concluded that protein or proteins are involved in 4-hydroxyisoleucine
mechanism do not have a long life, may be due to rapid degradation
cycle or not abundantly available in normal condition within the cells
105
which need to be synthesised newly. This observation also suggests that
there are different mediators are involved in isoleucine mechanism of
action from 4hydroxyisoleucine.
Figure 4.5. The effect of cycloheximide (CHX) on glucose uptake and ATP
content in BRIN-BD 11 cells. BRIN-BD11cells incubated with 500 μM 4-
hydroxyisoleucine and isoleucine in the presence of 0.8 μM cycloheximide (CHX).
There is a significant changes in glucose consumption between 4-hydroxyisoleucine
and 4-hydroxyisoleucine control group. There is significant increase in glucose
consumption observed in isoleucine group (p<0.01). There is no changes in ATP level
in all groups.
106
4.6 Wortmannin and PI3Kinase role in 4-hydroxyisoleucine and
isoleucine activities in BRIN-BD11 cells
Broca et al. (2004) showed that 4-hydroxyisoleucine can increase
PI3Kinase activity in both muscle and liver independently of insulin in
the presence of high glucose concentration, and the study by Jaiswal et
al. (2012) confirmed that adding wortmannin, a potent inhibitor of PI
3Kinase, inhibits the 4-hydroxyisoleucine-induced GLUT4
translocation and glucose uptake in muscle. These studies suggest a
key role for PI 3Kinase in mediating the effects of 4-
hydroxyisoleucine. Adding 5nM of wortmannin (IC50 = 2 - 4 nM) to
the culture medium of BRIN-BD11 at the same time with a 4-
hydroxyisoleucine and isoleucine and incubated for 24 hours did not
inhibit 4-hydroxyisoleucine increasing effect on glucose consumption
(Fig 4.6). It was interestingly observed that wortmannin blocks
isoleucine effect even glucose consumption significantly reduces in
front of wortmannin compared to isoleucine control
group (Fig 4.6). It suggested that the PI3Kinase plays role in isoleucine
effects on increasing glucose uptake but not in 4-hydroxyisoleucine
in BRIN-BD 11 cells. It seems that inhibiting PI3Kinase activity in
BRIN BD 11 cells does not inhibit 4-hydroxyisoleucine action contrary
to previous findings in muscles reported by Jaiswal et al. It can be
concluded that there is a strong relation between the mechanism of
hypoglycaemic action of isoleucine and PI3 Kinase activity as
reported by Doi et al in 2003.
107
Figure 4.6. Glucose consumption in BRIN-BD11 cell was not affected in 4-
hydroxyisoleucine group in front of 5 nM wortmannin after 24 hours. There is a
significant reduction in glucose consumption in the isoleucine group in front of
wortmannin (p<0.01).
108
4.7 Wortmannin and Cycloheximide (CHX) increase glucose
consumption in BRIN-BD11 cells
It was noticeable in our several runs with wortmannin and CHX that
glucose consumption in experiments with both compounds was
significantly higher individually compared to the control without any
compound (Fig 4.6). To verify it we set up an independent trial to
investigate the effect of 10 nM and 5 nM wortmannin and 0.8 μM CHX
on BRIN-BD11 cell's glucose consumption. We used both cell count and
protein assay together for normalisation to reduce the bias as much as
possible. The results confirmed our previous findings, which both
wortmannin and CHX significantly increased glucose consumption in
BRIN-BD 11 cells incubated with RPMI-1640 medium contains 22mM
glucose after 24 hours (Fig 4.7).
Figure 4.7. BRIN-BD11 cells were incubated with 5 nM and 10 nM wortmannin (W)
and 0.8 μM CHX in RPMI-1640 with 22 mM glucose for 24 hours. Glucose
measurements show a very significant increase in glucose consumption in both
wortmannin and CHX groups (p<0.01). No significant difference was observed
between 5 nM and 10 nM wortmannin group (ns).
109
The same finding has been observed in rat epididymal fat pad exposed to
0.8 mM (1 microg/ml) CHX and there is no clear explanation for it
(García-Sáinz et al. 1977). Current study shows that CHX produces same
effect on beta cell with unknown mechanism. It also clarifies that
increase of glucose uptake baseline in all CHX groups respect to non-
CHX groups are due to the CHX stimulatory effect on glucose uptake
and adding 4-hydroxyisoleucine to the cells before CHX unlike
isoleucine does not stimulate the glucose uptake further than CHX
control group baseline.
Current study finding about the effect of wortmannin on increasing
glucose uptake in beta cell is new as there is no such a report found in
the literatures. It was shown in previous works that wortmannin as
inhibitor of PI3Kinase, amplifies glucose-stimulated insulin secretion in
beta cells (Zawalich et al. 2002) and it has also suggested that
wortmannin induces insulin secretion in high glucose concentration (16.7
mM) by inhibiting phosphodiesterase to increase cAMP content which
indicates that PI3-kinase inhibits insulin secretion by activating
phosphodiesterase to reduce cAMP content (Nunoi et al. 2000).
Considering wortmannin stimulatory effect on glucose uptake and
glucose-dependent insulin secretion hypothesizes that inhibition of
PI3Kinase may activates parallel compensatory mechanisms within the
beta cell which results in a glucose influx into the cell and insulin
secretion respectively. It creates unique research opportunity to address
this finding.
110
4.8 The mTOR role in 4-hydroxyisoleucine and isoleucine
mechanism of actions
PI3kinase plays an essential role in insulin-stimulated glucose uptake,
GLUT 1 and GLUT 4 translocations in adipose and other peripheral
tissues responsible for glucose disposal which is blocked completely by
wortmannin (Okada et al. 1994, Clarke et al. 1994). mTOR (mammalian
Target of Rapamycin) as downstream molecules in PI3Kinase-AKT
(protein kinase B) pathway is a critical cellular regulator. The mTOR
signalling pathway integrates a large number of environmental changes
and cellular stresses, including reactive oxygen species, hyperosmotic
and nutrient depletion stress as well as growth factor and cytokine
signalling (Corradetti et al. 2006). Previous studies showed that mTOR
enhances the intrinsic activity of GLUT1 transporters (Wieman et al.
2007), GLUT 1 expression and glucose uptake (Buller et al. 2008). It
was observed that isoleucine stimulates insulin-independent glucose
uptake mediated by activating PI3Kinase and independent of activation
of mammalian target of rapamycin (mTOR) like leucine which is a
potent activator of mTOR (Doi et al. 2003, Zhang et al. 2007). In
previous experiments it was determined that PI3 Kinase activity is
directly involved in both isoleucine and 4-hydroxyisoleucine ability in
stimulating glucose uptake in the beta cell. Considering the role of
mTOR in PI3 Kinase signalling pathway and glucose uptake, the effect
of isoleucine and 4-hydroxyisoleucine were studied in the presence of 10
nM KU 0063794, a selective inhibitor of mammalian target of rapamycin
(mTOR) (IC50 =10 nM for mTORC1 and mTORC2) without any
activity at PI3-kinase or 76 other kinases within the cells (García-
Martínez et al. 2009).
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Results revealed that inhibition of mTOR solely with a specific inhibitor
does not change the effect of isoleucine and 4-hydroxyisoleucine on
increasing glucose consumption in beta cells (Fig 4.8) which reiterate the
Doi's findings about isoleucine and extends it to 4-hydroxyisoleucine.
Persistent results in each set of experiments by KU 0063794 determine
that mTOR does not play essential role in the mechanisms of action of
isoleucine and 4-hydroxyisoleucine.
Figure 4.8. The effect of mTOR inhibitor on glucose consumption rate of 4-
hydroxyisoleucine and isoleucine in BRIN-BD11 cell. mTOR inhibition does not
change the glucose uptake in BRIN-BD11 cells indicates that mTOR does not play
main role in uptaking glucose by pancreatic beta cells. There are also no changes in
the activity of isoleucine and 4-hydroxyisoleucine in the presence of 10 nM KU
0063794, a selective mTOR inhibitor. Isoleucine and 4-hydroxyisoleucine
significantly increased glucose consumption in BRIN-BD11 cells incubated with
RPMI-1640 with 22 mM glucose (p<0.01).
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PI3 Kinase-AKT-mTOR pathway plays important role in glucose uptake
regulation as one of the major downstream intracellular signalling
pathways in response to binding of many substances like growth factor
and insulin to their receptors on the surface of the cell. Activation of PI3
Kinase-AKT-mTOR pathway increases glucose uptake via interacting
with GLUT 1 and in less extent GLUT 3 expression and activity through
mTOR (Fig 4.9) (Bryan et al. 2005). GLUT1 plays critical role in
maintaining basal glucose uptake required to sustain cellular respiration
in all cells. Expression levels of GLUT1 in cell membranes are increased
by reducing glucose levels and decreased by increased glucose levels.
GLUT1 is able to compensate for GLUT2 deficiency or under expression
in beta cells and maintains an appropriate rate of glucose uptake to
sustain glucose metabolism in pancreatic beta-cells (Liang et al. 1997).
113
Figure 4.9. AKT and mTOR are activated by membrane receptors and stimulate
glycolysis in part by AKT-induced localization of the glucose transporters, including
GLUT1 and GLUT3, to the cell surface, and maintenance of hexokinase function in
the absence of extrinsic factors with resultant glucose production. PI3K,
phosphatidylinositol-3-kinase. (Adapted from Bryan et al. 2005)
It drew the attention to investigate the role of the GLUT 1 in the effect of
isoleucine and 4-hydroxyisoleucine on glucose uptake considering that
the GLUT1 is the most abundant glucose transporters in the beta cell
which facilitates a main glucose influx into the cell.
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4.9 Role of GLUT1 in 4-hydroxyisoleucine and isoleucine mechanism
of actions in BRIN-BD11 cells
BRIN-BD11 cells were incubated 24 hours with isoelucine and 4-
hydroxyisoleucine in RPMI 1640 medium containing 22 mM glucose in
front of 5 μM STF 31, a cell-permeable sulfonamide that selectively
inhibits GLUT1 and glucose uptake (Chan et al. 2011) to achieve a
specific GLUT1 inhibiting status without compromising other
transporters in order to evaluate the role of GLUT1 in the effect of
isoleucine and 4-hydroxyisoleucine.
STF 31 was shown to inhibit the growth of VHL-deficient renal cell
carcinomas (RCCs) dose-dependently (5 μM) by directly targeting
GLUT1 without binding to other glucose transporters, and it does not
inhibit a broad range of 50 tested kinases (Chan et al. 2011). 4-
hydroxyisoleucine glucose consumption increasing effect in BRIN-BD11
cells reduced in front of STF 31 but GLUT1 inhibition did not show
any significant changes in the activity of isoleucine on glucose
consumption in BRIN-BD11 cells (Fig 4.10a). ATP measurements
confirmed a remarkable reduction in ATP content of the cell in STF 31
groups after 24 hours (Fig 4.10b). The findings determine that 4-
hydroxyisoleucine mechanism of action is profoundly dependent on
GLUT1 activity and it is in line with its mode of actions, insulin-
independent increase of basal glucose uptake in peripheral tissues as
GLUT1 is responsible for basal glucose uptake in cells.
The beta pancreatic cell membrane has a very high capacity of transporting
glucose into the cytoplasm compared to other cells. High capacity of
glucose transport in beta pancreatic cell is due to GLUT2 which has a
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very high Vmax and high Km of 15-20 mM (Guillam et al. 1997).
GLUT1 with low Km is also expressed in pancreatic beta cells. A
previous study showed thatGLUT1 expression is higher when glucose is
low, and GLUT2 expression is more profound when glucose is high in
the culture media (Tal et al. 1992). Incubation of BRIN-BD11 cell with
22 mM glucose and selective GLUT1 inhibitor, STF 31 for 24 hours
increases glucose uptake significantly, which implicates that GLUT1
inhibition may lead to over-expression of GLUT2 or shifting towards
other uptake mechanisms as a compensatory phenomena. Rabuazzo et
al in 1993 showed that inhibition of only GLUT1 when GLUT2 is
functioning in hamster-derived pancreatic beta cell line (HIT) enhances
insulin release. In our study, STF 31 increased glucose consumption,
which can be explained by referring to Rabuazz's findings as an insulin
release enhancement depends on higher rate of glucose transport which
means more glucose consumption. If GLUT1 inhibition enhances
insulin secretion in beta cell as shown in Rabuazzo study and insulin
secretion process consumes energy (ATP) and logically the ATP content
should drop after an increase prior to release. The end point
measurement of the cell ATP content after 24 hours incubated with STF
31 showed significant drop.
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Figure 4.10. Glucose consumption and ATP content in BRIN-BD11 cell in the
presence of STF 31. Glucose consumption was increased significantly (p<0.01) in
BRIN-BD11 cell in the presence of 5 μM of STF 31 after 24 hours incubation. The
glucose consumption increase was inhibited significantly in 4-hydroxyisoleucine
group in front of STF 31(p<0.01) but not in isoleucine group (a). The ATP content of
the cells decreased dramatically by STF 31 (p<0.001) and remained unchanged in all
STF 31 groups including isoleucine and 4-hydroxyisoleucine (b).
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GLUT1 inhibition dramatically interferes with 4-hydroxyisoleucine
activity as in all our previous findings the 4-hydroxyisoleucine had the
higher increasing effect on glucose consumption in BRIN-BD11 cell
compared to isoleucine but its effect reduced significantly in front of
STF 31. It can be hypothesized by reviewing current findings that
GLUT1 plays a major role in 4-hydroxyisoleucine mechanism of action
but not in isoleucine.
4.10 The role of calcium on the effects of 4-hydroxyisoleucine and
isoleucine on glucose consumption rate and ATP content in BRIN-
BD11 cells
The glucose transporter 1 (GLUT1) is expressed in a wide variety of cell
types and is largely responsible for maintaining the basal level of glucose
uptake. A previous study suggested that cytosolic calcium plays a role in
modulating basal glucose uptake and GLUT1 activity. It also showed
that lowering extracellular calcium decreases basal glucose uptake in rat
epithelial cells (Quintanilla et al. 2000). It was observed that intracellular
calcium stimulates glucose uptake in skeletal muscle cell and inhibition
of calcium release from the sarcoplasmic reticulum (SR) reduces sugar
uptake, whereas the increase of calcium release from SR stimulates
glucose transporter activity (Youn et al. 1991). A recent study using
shRNA-mediated silencing showed that the recently-identified
mitochondrial calcium uniporter (MCU) is essential for a sustained
glucose-induced increase in the cytosolic ATP/ADP ratio in pancreatic
beta cells (Tarasov et al. 2012). The mitochondrial calcium uniporter
(MCU) is located in the mitochondrial inner membrane that regulates
calcium uptake into mitochondria and plays a key role in mitochondrial
calcium homeostasis and cellular physiology. It regulates cytoplasmic
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calcium signals, cell bioenergetics and also glucose-dependent insulin
secretion in pancreatic beta-cells by regulating mitochondrial calcium
uptake (Alam et al. 2012, Mallilankaraman et al. 2012).
Considering the related available data for the role of calcium and MCU
as a mitochondrial calcium homeostasis regulator drew the attention to
evaluate the role of mitochondrial calcium uniport channels in isoleucine
and 4-hydroxyisoleucine effects in BRIN-BD 11 cell glucose uptake. To
study the calcium role, Ruthenium Red has been used. Ruthenium Red is
an inhibitor of mitochondrial calcium uniporter (MCU) which blocks
calcium uptake and release from mitochondria (Bernardi et al. 1984). It
also inhibits calcium release from ryanodine-sensitive intracellular stores
and blocks cell membrane-located capsaicin-activated cation channels
(Bernardi et al. 1984, Szallasi et al. 1999, Israelson et al. 2008). It was
shown that ruthenium red inhibits neurotransmitter release by blocking
voltage-sensitive Ca2+ channels (Xu L et al. 1999). Ruthenium red
efficiently blocks the L-type calcium channel in a dose-dependent
manner which reaches an inhibition of 85% at 5 µM (Malécot et al.
1998).
To investigate the role of calcium in the modulation of glucose uptake in
BRIN-BD11 cells by isoleucine and 4-hydroxyisoleucine, cells were
treated with 5 µM of ruthenium red simultaneously with 500 µM
isoleucine and 4-hydroxyisoleucine leaded to very significant decrease
in glucose consumption in 4-hydroxyisoleucine group and inhibition of
the glucose consumption increase in isoleucine groups (Fig 4.11a).
Several experiment repeats with five replicates in each set, showed a
persistent result which implicates the roles of calcium in both isoleucine
and 4-hydroxyisoleucine mechanisms of action. Inhibition of
mitochondrial calcium uniporter (MCU), calcium channels and calcium
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signalling by ruthenium red not only inhibits 4-hydroxyisoleucine
activity but creates a reverse activity in 4-hydroxyisoleucine. The
glucose consumption decreased significantly in 4-hydroxyisoleucine
group treated with ruthenium red compared to controls (Fig 4.11a).
Contrary to 4-hydroxyisoleucine group, glucose uptake remarkably
increased in isoleucine group treated by ruthenium red compared to
controls(Fig 4.11a). It seems that intracellular calcium signalling does
not play an equal role in 4-hydroxyisoleucine and isoleucine
mechanism of actions. It may be explained by referring to the previous
findings about the relation of calcium and glucose uptake that inhibition
of cytoplasmic calcium channels by ruthenim red alongside with MCUs
result in decrease of cytoplasmic calcium due to reducing the diffusion
of extracellular calcium (Bernardi et al. 1984, Szallasi et al. 1999,
Israelson et al. 2008). Reducing cytoplasmic calcium triggers the
calcium release from sarcoplasmic reticulum (SR) and stimulates glucose
transporter activity (Youn et al. 1991). We also observed that ruthenium
red is solely increasing glucose consumption in BRIN-BD11 cells (Fig
4.11a). The reduction in cellular ATP content after 24 hours in all
ruthenium red groups and more significantly in isoleucine plus
ruthenium red (Fig 4.11b) shows that inhibition of calcium channels
affects bioenergetic status of the cell and indicates important role of
calcium in both in isoleucine and 4-hydroxyisoleucine effects. BRIN-
BD11 cell's glucose uptake paradoxical behaviour in isoleucine and 4-
hydroxyisoleucine groups in the presence of ruthenium red determine a
distinct difference in 4-hydroxyisoleucine and isoleucine mechanisms of
action which is in line with our previous findings. The ruthenium red
neutralizes the glucose uptake stimulatory effect of 4-hydroxyisoleucine
but on the other hand it has synergy with isoleucine glucose uptake
activity.
120
a)
b)
Figure 4.11. The effect of ruthenium red on Isoelucine and 4-hydroxyisoleucin
activities in BRIN BD11 cells. The glucose consumption in the presence of 5 µM
ruthenium red in BRIN-BD11 cell in RPMI-1640 with 22 mM glucose after 24 hours
(a). Glucose consumption decreased very significantly in 4-hydroxyisoleucine group
treated simultaneously with ruthenium red (p<0.01) and there is no sign of inhibition
in isoleucine group. Isoleucine showed higher glucose consumption in front of
121
ruthenium red compared to isoleucine control without ruthenium red (a) which shows
paradoxical behaviour of 4-hydroxyisoleucine in combination with ruthenium red on
glucose uptake. Ruthenium red is solely increasesing glucose consumption
significantly (p<0.01) (a). The ATP concentration of cells at the end of the
experiment shows a significant reduction in ATP content in all ruthenium red treated
groups (p<0.01) and with greater decreases in isoleucine group (b) Non treatment
groups did not show any significant difference in ATP content (c).
The intracellular calcium level is regulated by the sarcoplasmic
reticulum (SR) as a reservoir of calcium, mitochondria and cytoplasmic
membrane calcium channels. Intracellular calcium uptake into the SR
regulates by the sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA 2a)
calcium pump and remaining of cytoplasmic calcium is removed via the
Na+/Ca2+ exchanger (NCX) or taken up into mitochondria by the
mitochondrial calcium uniporter (MCU) (Bodi et al. 2005, Cingolani et
al. 2007). Mitochondrial oxidative phosphorylation as a source of ATP
production is dependent on mitochondrial calcium uptake via a
ruthenium red-sensitive mitochondrial calcium uniporter (MCU). The
MCU calcium transport rate is slow and its affinity for calcium is low
(Km 10-20 mM). The Na+/Ca2+ exchanger is responsible for extruding
calcium from mitochondria (Denton et al. 1985,Gunter et al. 1990).
Calcium release from SR is controlled by ryanodine receptors (RyR)
and inositol triphosphate receptors (IP3R). Depletion of the SR calcium
reservoir is sensed by stromal interaction molecules (STIMs) located
on the SR membrane and monitor its calcium content. Sensing of the
low calcium content of SR by STIMs activates store-operated
calcium channels (SOCE) on the membrane leading to calcium influx
stimulation (Fig 4.12) (Mascia et al. 2012). G protein coupled receptor
activation will increase the release of calcium from SR through the
122
production of IP3, (Inositol 3 Phosphate) which binds to IP3 receptor on
the surface of SR (Mascia et al. 2012).
Considering cellular calcium homeostasis shows that ruthenium red
reduces calcium release from SR in pancreatic beta cells by inhibiting
ryanodine receptors (RyR), calcium influx by blocking calcium
channels on cytoplasmic membrane and mitochondrial calcium
uptake by inhibiting MCUs which lead to intracellular calcium
depletion. The cellular protective mechanism will try to compensate
intracellular calcium depletion via increasing calcium release from the
SR by activating IP3 receptors (parallel gate which is not blocked by
ruthenium red) through triggering production of IP3 by intrinsic
enzymatic activity, reducing calcium uptake by sarcoplasmic
reticulum Ca2+-ATPase 2a (SERCA2) and stimulation of different
types of membrane calcium channel to increase calcium influx.
Activation of parallel unblocked calcium gates on SR leads to
depletion of SR calcium and activation of stromal interaction
molecules (STIMs) as SR calcium sensor. Stromal interaction
molecules (STIMs) stimulate calcium influx as well. Other important
pumps could help the cell to compensate the lack of cytosolic calcium
in such a condition is a sodium-calcium exchanger (NCX).
The sodium-calcium exchanger (NCX) is a high
capacity antiporter membrane protein uses the electrochemical
gradient energy, allowing sodium to flow down its gradient across
the plasma membrane in exchange for the counter-transport
of calcium ions. The NCX is important in cellular calcium
homeostasis expresses in many different cell types and is capable of
exchanging a single calcium ion with three sodium ions in both ways
depend on a gradient (Yu SP et al. 1997, Dipolo et al. 2006).
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Figure 4.12. Integration of the calcium signaling circuitry. The major regulators of
calcium homeostasis in keratinocytes are depicted. Plasma membrane pumps and
channels (PMCA, NCX, and SOCE) regulate flux in and out of the cytosol. G-
protein–coupled receptors (calcium-sensing receptor (CaR) and others not shown)
initiate signals that modify compartmentalized calcium stores (e.g., IP3). Calcium
ATPases on organelles (SERCA, SPCA1) monitor and replenish intracellular storage
sites. CCa2+, calcium; DAG, diacylglycerol; ER, endoplasmic reticulum; IP3R,
inositol 1,4,5-trisphosphate receptor; NCX, Na+/Ca2+ exchanger; NFAT, nuclear
factor of activated T cells; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein
kinase C; PLC, phospholipase C; PMCA, plasma membrane Ca2+ ATPase; SERCA,
sarco (endo)plasmic recticulum Ca2+ ATPase; SOCE, store-operated calcium
channels; STIM, stromal interaction molecule; TRPC, transient receptor potential C.
The figure is modified from Savignac et al. (2011) with permission from Elsevier.
(Completely adapted from Mascia et al . 2012)
All these pathways for balancing disturbed calcium homeostasis need
energy and ATP production may slow down due to MCU blockage by
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ruthenium red (Fig 4.11b). To compensate for slow energy
production, cells attempt to take up more fuel by increasing glucose
uptake to boost up energy production in mitochondria. On the other
hand increasing glucose uptake (Fig 4.11a) itself leads to beta cell
membrane depolarization and subsequent calcium influx (Srinivasan
et al. 2002). Ruthenium red indirectly increases glucose uptake via
disturbing intracellular calcium homeostasis. Previous animal trials
showed that verapamil, a calcium channel blocker which blocks L-
type calcium channels mainly and prevents cellular calcium influx,
reduces blood glucose level and increased insulin levels in STZ-
induced diabetic mice by increasing glucose uptake in beta cells and
enhancing pancreatic beta cell function (Xu G et al. 2012).
Mitochondrial calcium uniporters (MCUs) and calcium roles in 4-
hydroxyisoleucine and isoleucine effects attracted the attention by
observing the effect of ruthenium red as an MCU inhibitor on isoleucine
and 4-hydroxyisoleucine activity. Does MCUs activator alter isoleucine
and 4-hydroxyisoleucine effects on glucose consumption, If it is assumed
based on current findings that MCUs inhibition can eliminate the effect
of the 4-hydroxyisoleucine. To answer the question, BRIN-BD 11 cells
were incubated with 500 µM isoleucine and 4-hydroxyisoleucine for 24
hours in the presence of 20 µM of kaempferol, a naturally occurring
flavonoid found in Gingko biloba and red wines that activates the
mitochondrial calcium uniporters (MCUs) (EC50 = 7 μM) (Montero et
al. 2004, Vay et al. 2007).
Kaempferol is able to increase glucose uptake in 3T3-L1 adipocytes and
act as an anti-diabetic agent via multiple targets such as acting as a
partial agonist of PPARgamma (Fang et al. 2008). It was also observed
125
that Kaempferol metabolite, kaempferol 3-neohesperidoside, is capable
to increase glucose uptake in myocytes mediated by PI3Kinase and
protein kinase C (PKC) pathways (Zanatta et al. 2008). Glucose
consumption in BRIN BD11 cells increased significantly after 24 hours
incubation with 20 µM of kaempferol without noticeable changes in
cellular ATP contents at the end of the experiment (Fig 4.13a,b). This
finding is in line with previous studies mentioned above and confirms its
glucose uptake stimulatory property. Kaemferol produced the similar
amount of increase in glucose consumption as isoleucine alone, but
significantly lower than the 4-hydroxyisoleucine which shows that 4-
hydroxyisoleucine stimulates the glucose uptake in much greater extent
to the kaempferol and isoleucine.
126
a)
b)
Figure 4.13. The effect of kaempferol on glucose consumption in BRIN-
BD11cells. Glucose consumption was significantly increased by 20 µM of
kaempferol (p<0.01) and dominantly in both isoleucine and 4-hydroxyisoleucine
groups incubated simultaneously with kaempferol (p<0.001) (a). A synergistic effect
of kaempferol with 4-hydroxyisoleucine and in greater extent with isoleucine has
been observed (a). ATP content of cells does not change in the presence of
kaempferol and kaempferol plus isoleucine but a noticeable decrease was observed in
4- hydroxyisoleucine plus Kaempferol group (p<0.05) (b).
127
Incubation of BRIN BD11 cells with 4-hydroxyisoleucine and isoleucine
alongside with 20 μM kaempferol in the same time for 24 hours showed
a remarkable increase in glucose consumption in both 4-
hydroxyisoleucine and isoleucine groups with kaempferol compared to
controls (Fig 4.13a). Interestingly, the glucose consumption in isoleucine
with Kaempferol group increased very significantly by more than 3 mM
compared to isoleucine alone (p<0.001). In all our previous findings, 4-
hydroxyisoleucine solely created greater effect than isoleucine but the
combination of isoleucine and kaempferol could create the equal effect
as 4-hydroxyisoleucine with or without kaempferol. These findings
indicate a strong synergistic effect between kaempferol and both
4hydroxyisoleucine and isoleucine on stimulating glucose uptake in
BRIN BD 11 cells and also demonstrate the role of calcium and MCU in
mechanisms of action of 4-hydroxyisoleucine and isoleucine. The results
also show the important role of calcium and mitochondrial calcium
uniporters in glucose uptake mechanism. ATP measurements did not
show changes in ATP content between kaempferol and isoleucine plus
kaempferol group but a significant decrease were observed in ATP
content of BRIN-BD 11 cells in 4-hydroxyisoleucine plus kaempferol
group (Fig 4.13b).
A previous gene expression study using microarray and real-time PCR
identified a set of metabolically relevant genes were induced by 20 μM
kaempferol after 24 hours including peroxisome proliferator–activated
receptor coactivator-1, carnitine palmitoyl transferase-1, mitochondrial
transcription factor 1, citrate synthase, and uncoupling protein-3 (da-
Silva et al. 2007). It is also shown that kaempferol increases the oxygen
consumption and cellular energy expenditure via increasing cyclic AMP
in skeletal muscle (da-Silva et al. 2007). Synergistic effect between
isoleucine, 4-hydroxyisoleucine and kaempferol suggests that the
128
previously identified metabolically relevant genes induced by
kaempferol in da-Silva et al study could have direct or indirect roles in
the mechanisms of action of 4-hydroxyisoleucine and isoleucine which
create a new interesting area of further investigation.
The results from ruthenium red and kaempferol experiments support
that intracellular calcium signalling and mitochondrial calcium
uniporters (MCUs) play an important role in 4-hydroxyisoleucine and
isoleucine glucose uptake stimulatory effect but their roles are not
similar in isoleucine and 4-hydroxyisoleucine mechanisms of action.
Analysing data from the effects of ruthenium red (Fig 4.11) as an MCU
and calcium signalling inhibitor and kaempferol as an MCU activator
(Fig 4.13) on isoleucine and 4-hydroxyisoleucine activity in BRIN-BD
11 cells indicate:
• Interference with calcium signalling and mitochondrial calcium
uniporter (MCU) using inhibitor, inhibits 4-hydroxyisoleucine
effect on increasing glucose consumption, but not the effect of
isoleucine whereas using MCU activator enhances both 4-
hydroxyisoleucine and in greater degree isoleucine effects which
means the important roles of calcium signalling and MCU in 4-
hydroxyisoleucine and isoleucine mechanisms of action.
• Isoleucine activity exhibits a significant increase in the presence of
both MCU activator and inhibitors , which is different from 4-
hydroxyisoleucine behaviour. Unlike isoleucine, inhibition of 4-
hydroxyisoleucine effect on stimulating glucose uptake in the
presence of ruthenium red indicates that calcium signalling is an
129
essential for its mechanism of action. It also reiterates that
calcium's role in the mechanism of action of 4-hydroxyisoleucine
is different from isoleucine.
• These findings support that different mechanisms and pathways
are involved in actions of isoleucine and 4-hydroxyisoleucine
apart from some similarities in their effects and molecular
structures.
4.11 The effect of the UK-5099 as a mitochondrial pyruvate carrier
(MPC) inhibitor on 4-hydroxyisoleucine and isoleucine glucose
utilisation in BRIN-BD11 cells
The results of previous experiments with ruthenium red and kaempferol
demonstrated the role of calcium and in a broader spectrum, the
mitochondria in 4-hydroxyisoleucine and isoleucine effects on glucose
uptake in BRIN-BD 11 cells. We also observed in all the previous
experiments that the ATP content of the cells using end point cell ATP
content measurement at the end of each experiment did not change
considering the increase in glucose consumption induced by 4-
hydroxyisoleucine and isoleucine. The BRIN-BD 11 cell is a beta
pancreatic cell which consumes glucose to make energy in the form of
ATP for its biological activities unlike adipose cell which can store
excess glucose by transforming it to triglyceride. As the glucose uptake
is an energy consuming activity and considering the previous
observations in the current study showing the glucose consumption
increases in front of 4-hydroxyisoleucine and isoleucine while the ATP
content unchanged , suggest that energy production process in
130
mitochondria is essential for 4-hydroxyisoleucine and isoleucine effects.
To evaluate the role of mitochondria and energy production cycle in the
effect of isoleucine and 4-hydroxyisoleucine, we designed an experiment
to assess 4-hydroxyisoleucine and isoleucine effects on glucose
consumption in BRIN-BD 11 cells in front of a mitochondrial pyruvate
carrier (MPC) blocker. Pyruvate is the end product of glycolysis and a
major substrate for the tricarboxylic acid (TCA) cycle in mitochondria
for producing ATP. Pyruvate is produced in cytosol and across the inner
mitochondrial membrane via MPCs which are located in inner
mitochondrial membrane (Herzig et al. 2012). Mitochondrial pyruvate
carrier (MPC) is essential for producing ATP.
The BRIN-BD 11 cells were incubated with 500 µM 4-
hydroxyisoleucine and isoleucine simultaneously with 35 µM 0f UK-
5099, an a-cyanocinnamate analogues which acts as specific and potent
inhibitors of mitochondrial pyruvate carrier (MPC) activity (Halestrap.
1978). A recent study by Divakaruni et al showed that UK 5099
increases glucose uptake in myocytes which directly proportional to the
extent of respiratory inhibition by UK 5099 but our study results
indicated that UK 5099 did not change the glucose consumption in
BRIN-BD 11 while the ATP content dramatically decreased after 24
hours incubation (Fig 4.14). This finding is not in line with Divakaruni et
al study. The difference between the biology of beta pancreatic cell and
myocytes which used in Divakaruni study could explain different
behaviours. On the other hand we expected to see a significant reduction
in glucose consumption in the UK 5099 control group as intervening
with ATP production cycle should reduce glucose uptake due to cellular
energy depletion but the glucose consumption in the UK 5099 groups
remained unchanged compared to no treatment control. However we did
not detect an increase in glucose uptake in the UK5099 group same as
131
Divakaruni observed in his study in myocytes but unchanged glucose
consumption in BRIN-BD 11 in front of the UK 5099 shows that
mitochondrial respiratory inhibition in beta cell may activate a feedback
to push the cell to take up more glucose for compensating the disrupted
energy production cycle.
Interestingly isoleucine and 4-hydroxyisoleucine effects were diminished
significantly in front of the UK 5099 (Fig 4.14) compared to the
controls. The results indicate that mitochondria and ATP production
cycle within the mitochondria are essential for both 4-hydroxyisoleucine
and isoleucine mechanisms of action. It raises a question for further
study about the effect of isoleucine and 4-hydroxyisleucine on
mitochondrial activity and glycolysis pathway in the cell. Theoretically
based on these findings, it is expected that the mitochondrial activity
may increase in front of 4-hydroxyisoleucine and isoleucine to drive the
cells towards more glucose uptake then it could justify why
mitochondrial pyruvate carrier inhibition blocks 4-hydroxyisoleucine and
isoleucine effects. It is very important to investigate the effect of 4-
hydroxyisoleucine and isoleucine on mitochondrial respiration because
of the essential role of mitochondria in diabetes and insulin resistance.
132
a)
b)
Figure 4.14. The effect of UK 5099 on glucose consumption in BRIN-BD 11 cells.
There is no significant change in glucose consumption in BRIN-BD 11 cell after 24
hours incubation with 35 µM UK 5099 (a). Glucose consumption shows a dramatic
decrease in both isoleucine and 4-hydroxyisoleucine in front of UK 5099 (p<0.01)
compared to UK 5099 and no treatment control groups (a). ATP level significantly
reduced in all UK 5099 groups (p<0.01) without difference between isoleucine and
4-hydroxyisoleucine groups (b).
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4.12 Analysis of the effects of 4-hydroxyisoleucine and isoleucine on
mitochondrial respiration in BRIN-BD 11 cells with Seahorse XF-24
autoanalyser
We used the Seahorse cell metabolism analyser to evaluate the
mitochondrial activity and glycolysis within the BRIN-BD 11 cells after
24 hours incubation with 500 µM isoleucine and 4-hydroxyisoleucine.
Seahorse XF (Extracellular Flux) analyser is the platform for metabolic
assays which simultaneously and in real time measures the two major
energy producing pathways of the cell, mitochondrial respiration and
glycolysis, in a microplate. The seahorse XF autoanalyser measures
oxygen consumption rate (OCR) as the indicator of mitochondrial
respiration and extracellular acidification rate (ECAR) which is
predominately the result of glycolysis at intervals of approximately 2-5
minutes. We measured the oxygen consumption rate (OCR) and
extracellular acidification rate (ECAR) in BRIN-BD11 cells after 24
incubation with isoleucine and 4-hydroxyisoleucine for 30 minutes in
three replicates for each group to study changes in the activity of
mitochondria and glycolysis. Results after normalisation using cell
counts showed that oxygen consumption rate (OCR) in BRIN-BD 11
cells was significantly higher in cells incubated with isoleucine and 4-
hydroxyisoleucine compared to control (Fig 4.15a). The OCR was
significantly higher in 4-hydroxyisoleucine group compared to
isoleucine group (Fig 4.15a,b). Increasing in OCR indicates that both 4-
hydroxyisoleucine and isoleucine increase the mitochondrial activity, but
4-hydroxyisoleucine effect on mitochondrial metabolism is much greater
than isoleucine in BRIN-BD11 cells. Extracellular acidification rate
(ECAR) measurement showed a significant rise in 4-hydroxyisoleucine
group, but not in isoleucine group (Fig 4.15c) which indicates the higher
134
glycolysis rate in the cells incubated with 4-hydroxyisoleucine compared
to isoleucine. The OCR/ECAR ratio is significantly higher in both
isoleucine and 4-hydroxyisoleucine groups compared to control (Fig
4.15d) with no significant difference between the 4-hydroxyisoleucine
and isoleucine groups. Seahorse study revealed that 4-hydroxyisoleucine
increases both mitochondrial metabolism rates shown as OCR and
glycolysis rate shown as ECAR but isoleucine only increases
mitochondrial metabolism rate without changing the glycolysis rate
significantly. These findings explain the inhibitory effect of the UK5099
as an MPC blocker on both 4-hydroxyisoleucine and isoleucine activities
in BRIN-BD 11 cells. It also indicates that their activities firstly
dependent on mitochondrial activity and secondly are not limited to
glucose uptake increase but they can increase cell metabolism. There is
possibility that increasing glucose uptake by 4-hydroxyisoleucine and
isoleucine is not a direct action due to increased glucose transporters or
glucose uptake pathways within the cell, but it could be the indirect
result of increasing the cell basal metabolism in the deepest layer. One of
the important common underlying pathophysiology in both diabetes type
1 and 2 is cellular metabolic defect due to lack of insulin and insulin
resistance, resulted in diabetic metabolic complications. Considering the
facts that our previous animal study showed a comprehensive modifying
effect of 4-hydroxyisoleucine on different metabolic parameter such as
blood sugar, triglyceride, cholesterol in diabetes type 1 models without
changing the level of insulin, and the role of calcium signalling specially
mitochondria calcium transporters, support the idea that 4-
hydroxyisoleucine and in lesser extent with probably different
mechanism isoleucine exert their anti-diabetic properties through
increasing cellular metabolism. Theoretically, if the metabolism of cell
increases, it needs to produce more energy and accordingly it requires to
135
uptake more glucose from the peripheral. It means that facilitating the
glucose uptake or increasing insulin sensitivity as suggested in some
previous literatures may not be a direct mechanism of 4-
hydroxyisoleucine's anti-diabetes effect like any available anti-diabetic
medications but they could be the results of its effect.
Seahorse measurement findings showed a lower glycolysis rate in
isoleucine compared to 4-hydroxyisoleucine which could support the
previous findings of the current study in the sense that 4-
hydroxyisoleucine and isoleucine mechanisms of action are not similar
in the cellular level and interacts with wider targets and pathways within
the cell leading to facilitate glucose utilisation probably via enhancing
glycolysis beside other mechanisms.
136
a)
b)
137
c) d)
Figure 4.15. Seahorse XF-24 autoanalyser results. Oxygen consumption rate
(OCR) in BRIN-BD11 cells increased very significantly after 24 hours incubation
with 500 µM isoleucine and 4-hydroxyisoleucine (p<0.001) (a). OCR measurement
indicated much greater increase in mitochondrial metabolism in 4-hydroxyisoleucine
groups in comparison with isoleucine groups (p<0.01) (a,b). Extra cellular
acidification rate as an indicator of glycolysis increased significantly in 4-
hydroxyisoleucine groups (p<0.05) but not in isoleucine groups (c). A non
statistically significant reduction in ECAR has been observed in isoleucine groups
(c). Average OCR/ECAR ratio shows significant increase in metabolic rate in cells
incubated with isoleucine and 4-hydroxyisoleucine (d). Average OCR/ECAR ratio is
lower in 4-hydroxyisoleucine because of increase in both ECAR and OCR which is
not observed in isoleucine (d).
138
Chapter V
Conclusions
139
We tried to create a diabetic rat model as close as possible to diabetes
type 1 condition with high blood glucose level and significant reduced
plasma insulin and insulin secretion capability of pancreatic beta cell
using a multiple intraperitoneal streptozotocine injection. Multiple dose
of streptozotocine injection for creating a stable type 1 diabetes animal
model is a well established and affordable technique which leads to a
significant reduction in pancreatic beat cells number and concomitant
reduction in insulin secretion capacity (King AJ. 2012). There is always
a limitation to create a right animal model, but our diabetic animal model
exhibits all expected metabolic complications of diabetes, including high
blood glucose, triglyceride, cholesterol and uric acid with noticeable
reduction in insulin compared to non diabetic animals. Treatment of
diabetic animals with 4-hydroxyisoleucine for four weeks modified the
metabolic complications of diabetes without changing insulin level
compared to the diabetic control group. Our study findings follow the
same results from previous studies with different animal models close to
type 2 diabetes (Broca et al. 2004, Sauvaire et al. 1998, Singh et al. 2010
and Haeri et al. 2009). It shows that 4-hydroxyisoleucine is not
necessarily dependent on insulin for exhibiting its anti-diabetic
properties as concluded in previous works. 4-hydroxyisoleucine could
increase glucose uptake without increasing insulin, otherwise in diabetes
type 2 animal models with inulinemia in Broca's study we should see
more sever insulinemia instead of significant reduction in insulinemia. It
also sheds a light on the fact that 4-hydroxyisoleucine effect is not
limited to insulin and it could exert more profound effect in utilizing
extra glucose and also modifies metabolic pathways.
We chose a pancreatic beta cell as a model to investigate the 4-
hydroxyisoleucine for further clarification of our animal study findings.
Studying the 4-hydroxyisolecine effect on pancreatic beta cell which
140
does not exhibit insulin induced glucose uptake pathway allowed us
assess its effect without insulin interference. Isoleucine was chosen as a
positive control because of its close molecular and anti-diabetic
properties to 4-hydroxyisoleucine. Incubation of BRIN-BD 11 cells as a
pancreatic beta cell model in a medium with 22mM glucose
concentration and 4-hydroxyisoleucine and isoleucine simultaneously for
24 hourse did not show any significant increase of insulin secretion
compared to control despite the significant increase in glucose
consumption. This finding was in line with our previous animal study
with 4-hydroxyisoleucine showing its insulin independent glucose
lowering effect and confirms that 4-hydroxyisoleucine does not act as a
insulinotropic agent. Increase of glucose uptake in beta pancreatic cell
without changes in insulin secretion and decrease of blood glucose level
in our diabetic rat model without significant change of plasma insulin
level, indicate that the mechanism of action involves in 4-
hydroxyisoleucine is different from previously understood. All the
suggested mechanisms such as insulin sensitizing and stimulation of
insulin secretion by previous researchers are not a direct effect of 4-
hydroxyisoleucine but they could be the indirect effects which resulted
from the activity of 4-hydroxyisoleucine on other mechanisms within the
cells. It seems that both 4-hydroxyisoleucine and isoleucine utilize the
glucose consumption via modulating the cellular basal glucose
consumption because they are capable of stimulation of glucose uptake
in pancreatic beta cell which does not express GLUT 4. It means that the
increase in glucose uptake is handled by GLUT 1 or GLUT 2.
Considering the fact that GLUT 2 is mainly expressed in beta cells not
other peripheral cells and both 4-hydroxyisoleucine and isoleucine are
able to show a hypoglycaemic effect in animal models and peripheral
cells, such as adipose and muscle cells, GLUT 1 could be a potential
141
candidate for handling increased glucose uptake. It is also in line with
the role of GLUT 1 in maintaining basal glucose uptake required for a
sustain cellular respiration in most of the peripheral cells.
Endpoint measurements of ATP content of the BRIN-BD11 cells did not
show any significant changes between 4-hydroxyisoleucine, isoleucine
and control groups as well as an insulin level measurement in culture
medium after 24 hours. Study with different concentrations of glucose
(5, 11 and 22 mM) indicated a direct correlation between the increase of
glucose uptake, ATP content and insulin secretion (Fig 4.2,4.3) which
confirm the physiologically responsiveness of BRIN-BD 11 cell as
expected. It means that the cell's behaviour follows the physiological
pattern, increasing glucose concentration in medium as expected leads to
more glucose uptake and more ATP production, resulted in more insulin
secretion.
Endpoint measurement of cell's ATP contents in a specific point of time
is not a accurate and proper method to investigate the ATP production
within the cell. As ATP continuously recycles within the cell, we need
more accurate quantitative method to measure ATP in a real-time format.
It raises the question that how the cell utilizes the extra glucose, which
entered the cell. It would be a key point in mechanisms of action of both
4-hydroxyisoleucine and isoleucine, which requires more detailed and
sophisticated study.
Rendering these data suggest that unlike insulin, which increase
immediate glucose uptake via translocation of GLUT4 in peripheral
tissues, 4-hydroxyisoleucine and isoleucine may increase the cellular
basal glucose consumption by interacting directly with the cellular
metabolism and energy production cycle. It also could explain why their
hypoglycaemic effects appear in the long term not short treatment as it
requires more time that basal glucose consumption picks up.
142
Inhibiting a variety of pathways related to glucose uptake and cell
metabolism using selected inhibitors revealed that there are substantial
differences between isoleucine and 4-hydroxyisoleucine mechanisms of
action unlike their similar glucose uptake stimulation effect and anti-
diabetic properties which is greater in 4-hydroxyisoleucine (Fig 5.1).
Figure 5.1. Summary of the in-vitro studies, testing the effect of different inhibitors
and activator on 4-hydroxyisoleucine and isoleucine glucose uptake stimulation
activity.
143
As previous study showed that 4-hydroxyisoleucine effect was blocked
by protein synthesis inhibition using CHX (Jaiswal et al. 2012), we
tested the effect of CHX on isoleucine and 4-hydroxyisoleucine activity
in BRIN-BD 11. Adding CHX to BRIN-BD11 cells loaded
simultaneously with 4-hydroxyisoleucine and isoleucine revealed that
unlike isoleucine, 4-hydroxyisoleucine glucose uptake stimulatory effect
reduced significantly in front of protein synthesis inhibitor. It suggests
that intermediary protein or proteins are involved in 4-hydroxyisoleucine
mechanism of action but not isoleucine. It could add explanation to why
4-hydroxyisoleucine exhibits its hypoglycaemic effect in long term
treatment. 4-hydroxyisoleucine is dependent on the synthesis of specific
proteins and up-regulation of protein expression requires time. It requires
more investigation to study the effect of 4-hydroxyisoleucine on proteins
and genes expression in BRIN-BD 11 cell and other cell models such as
adipose and skeletal muscle. It could be an important subject for the
future research, providing more detailed insight about the mechanisms
are involved in 4-hydroxyisoleucin activity.
As explained in the beginning, GLUT 1 plays a major role in
maintaining the basal glucose uptake required to sustain cellular
respiration in all types of cell. GLUT 1 is the most abundant glucose
transporters in the beta cells and it is able to compensate for the GLUT 2
deficiency or under expression in beta pancreatic cells and maintain an
appropriate rate of glucose uptake to carry on glucose metabolism and
insulin secretion activity in pancreatic beta cells (Liang et al. 1997).
Partially blocking the GLUT 1 activity in BRIN-BD 11 cells with STF-
31 as a highly selective GLUT 1 inhibitor, showed a direct dependency
of 4-hydroxyisoleucine on GLUT 1 in comparison with isoleucine. The
direct correlation between GLUT 1 activity and 4-hydroxyisoleucine
glucose uptake stimulation, suggests that 4-hydroxyisoleucine directly or
144
indirectly increases cellular basal glucose uptake in long term and it may
increase GLUT 1 expression as well. It supports the notion that 4-
hydroxyisoleucine exhibits its activity in the long term treatment in
animal models and it does not show an immediate glucose lowering
effect like some anti-diabetic medications which increase insulin
mediated glucose uptake. Unfortunately, it was out of our available
resources to examine GLUT 1 expression in protein and gene level in
BRIN-BD 11 but it is recommended for the future research to study
GLUT 1 protein and gene expression in BRIN-BD 11 cell incubated with
4-hydroxyisoleucine in high glucose concentration. Based on our
findings, GLUT 1 plays a critical role, but only increasing GLUT 1
expression or activity do not completely explain all 4-
hydroxyisoleucine's anti-diabetic properties and other mechanisms are
involved.
GLUT 1 inhibition by STF-31 reduces the glucose uptake in 4-
hydroxyisoleucine group but not isoleucine group. On the other hand,
isoleucine effect was diminished by inhibiting PI3Kinase activity in
BRIN-BD11 cells with Wortmannin without significant change in 4-
hydroxyisoleucine activity. PI3Kinase plays a variety of roles in beta
pancreatic cells including increase of insulin gene transcription, cell
proliferation and suppress appotosis (Cerf. 2013). It was shown that
PI3Kinase inhibition in pancreatic beta cell, increased glucose uptake
and glucose-stimulated insulin secretion (Zawalich et al. 2002). Broca's
study in 2004 showed that 4-hydroxyisoleucine administration increased
PI3Kinase activity associated with IRS-1 in both muscle and liver same
as insulin but combining insulin and 4-hydroxyisoleucine did not
increase PI3Kinase activity further. Our finding does not support the
direct connection between 4-hydroxyisoleucine and PI3Kinase.
Increasing of PI3Kinase activity as detected in previous study could be
145
result of other pathways activation within the cell. In contrary with 4-
hydroxyisoleucine, our finding supports the role of PI3Kinase in
isoleucine activity. It is important to emphasis that our experiment only
provide a snap shot of interaction between 4-hydroxyisoleucine and
isoleucine with PI3Kinase and it requires more investigation to assess
the role of PI3Kinase in both 4-hydroxyisoleucine and isoleucine
mechanism of actions as PI3Kinase is involved in many pathways within
the cell.
It is an interesting finding of the opposite behaviour of 4-
hydroxyisoleucine and isoleucine in front of different inhibitors, despite
their similar effect on increasing the glucose uptake in BRIN-BD11 cells.
The different behaviours of isoleucine and 4-hydroxyisoleucine in front
of selected inhibitors clearly demonstrate a difference in mode of
actions between 4-hydroxyisoleucine and isoleucine.
Insulin independent increase of glucose uptake in BRIN-BD 11 cells
incubated by 4-hydroxyisoleucine and isoleucine raised a question about
the effect of these molecules on the cellular metabolism. As previously
explained, 4-hydroxyisoleucine seems to increase basal glucose uptake
in longer treatment in both in-vitro and in-vivo studies. Theoretically, It
requires that 4-hydroxyisoleucine facilitate the glucose utilization within
the cell via increasing cytoplasmic metabolism and mitochondrial
respiration.
The first step towards understanding the role of mitochondria was
assessed using mitochondrial calcium uniporter (MCU) inhibitor and
activator. It allows to study the role of calcium in 4-hydroxyisoleucine
and isoleucine mechanisms of action as well as mitochondria. The
increase of intracellular calcium and release of calcium from the
sarcoplasmic reticulum (SR), stimulate glucose uptake (Youn et al. 1991)
146
which shows the role of calcium in glucose uptake mechanisms. MCU
plays an essential role for a sustained glucose-induced increase in the
cytosolic ATP/ADP ratio in pancreatic beta cells (Trasov et al. 2012).
Partial inhibition of MCU in BRIN-BD 11 using ruthenium red created
an opposite effect in 4-hydroxyisoleucine and isolecuine groups. 4-
hydroxyisoleucine effect was diminished significantly, but isoleucine
showed some degree of synergistic effect with ruthenium red in
increasing glucose uptake. Both 4-ydroxyisoleucine and isoleucine
showed a significant increase in glucose uptake caused by activation of
MCU using kaempferol as an MCU activator. It does not only support
the crucial role of mitochondria and calcium in their mechanisms, but
shows different mechanisms are involved. It seems both 4-
hydroxyisoleucine and isoleucine interact with mitochondria but in via
seperate mechanisms. This experiment raises a strong idea to develop a
new research in the future to examine the role of calcium in 4-
hydroxyisoleucine and isoleucine activities which could be one of the
important aspects of 4-hydroxyyisoleucine and isoleucine mechanisms
of action.
Mitochondrial metabolism interruption in BRIN-BD11 cell using the
UK5099, a specific mitochondrial pyruvate carrier (MPC) inhibitor when
loaded simultaneously with 4-hydroxyisoleucine and isoleucine led to
significant inhibition of both 4-hydroxyisoleucine and isoleucine glucose
uptake stimulation effect. It proves the notion about the role of
mitochondrial respiration in their mode of actions. Real time metabolic
activity analysis of the BRIN-BD11cells after 24 hours incubation with
4-hydroxyisoleucine and isoleucine in the presence of 22mM glucose
with seahorse autoanalyser confirmed such a role of mitochondria. A
significant increase in metabolic activity was observed in cells after 24
147
hours incubation with 4 hydroxyisleucine and isoleucine but significantly
greater in 4-hydroxyisoleucine group. It explains the greater glucose
uptake in front of the same concentration of 4-hydroxyisoleucine
compared to isoleucine which was persistent throughout our
experiments. Reviewing the seahorse data shows a meaningful
difference between isoleucine and 4-hydroxyisleucine apart from their
effect on mitochondrial activity. Both oxygen consumption rate and
extracellular acidification rate were significantly increased by 4-
hydroxyisoleucine but no significant increase was detected in
extracellular acidification rate as the indication of glycolysis in
isoleucine group. 4-hydroxyisoleucine interacts with glycolysis cycle in
the cytoplasm as well as mitochondrial respiration but isoleucine
increases the mitochondria respiration without affecting the glycolysis
rate. It could explain the different behaviours of isoleucine and 4-
hydroxyisoleucine in front of some inhibitors in this study.
Seahorse autoanalyser results ignited the idea that there may be a link
between 4-hydroxyisoleucine activity and glucokinase. Glucokinase
requires a much higher glucose concentration for maximal activity.
Glucokinase is found in the liver and pancreatic beta cells and it plays an
important role in liver due to its high Vmax, allowing the liver to
effectively remove excess glucose, and minimize hyperglycaemia after
eating. Glucokinase unlike hexokinase which is inhibited by the product
of its reaction, is not inhibited by glucose 6 phosphate (G6P). Previous
studies showed that expression of glucokinase in cultured human muscle
cells treated with AdCMV-GKL results in proportional increases in
insulin-independent glucose disposal, and that muscle glucose storage
and utilization becomes controlled in a glucose concentration-dependent
manner in AdCMV-GKL-treated cells (Baqué et al. 1998). It can raise
the hypothesis that 4-hydroxyisoleucine may involve in facilitating
148
glucokinase activity as the main regulator of glycolysis alongside with
other possible mechanisms which may be in common with isoleucine
considering the facts that the hypoglycaemic activity of 4-
hydroxyisoleucine is seen in higher concentration of glucose. The
Seahorse autoanalyser results could support the idea of involvement of 4-
hydroxyisoleucine in the glucokinase pathway as it increases glycolysis
as well as mitochondrial respiration unlike isoleucine which does not
affect glycolysis. It will require more detailed studies to be done in the
future to investigate the interaction between glucokinase and 4-
hydroxyisoleucine. Unfortunately, we could not carry such study due to
limited resources and facilities, but it would be a great idea for the
further research project.
We can summarise the conclusions from animal and cellular studies
about 4-hydroxyisoleucine as follows:
• 4-hydroxyisoleucine has a unique long term anti-diabetic activity
as observed and it possibly could increase the cellular basal
glucose consumption in high glucose concentration over a longer
period of time without a significant immediate effect on glucose
uptake similar to insulin.
• 4-hydroxyisoleucine anti-diabetic effects are independent of
insulin but its effect on increasing basal glucose uptake in long
term treatment can contribute to the stronger insulin effect on
peripheral tissues. Insulin binding to its receptor, creates a surge in
basal glucose uptake by promoting GLUT 4 translocation to the
plasma membrane. If the basal glucose uptake increases, the same
amount of insulin can create a stronger effect on lowering glucose
149
level. Increased basal glucose uptake in peripheral cells via 4-
hydroxyisoleucine could explain the insulin-sensitizing property
of 4-hydroxyisoleucine in insulin resistant diabetic models studied
before.
• 4-hydroxyisoleucine modifies metabolic complications of
diabetes, most importantly dyslipidemia by decreasing
triglyceride, cholesterol, low density lipoprotein (LDL) levels in
blood. It also reduces plasma uric acid in diabetes as well as
hyperglycaemia and dyslipidemia. It is concluded that 4-
hydroxyisoleucine is not only a hypoglycaemic agent, but it is
capable of limiting the disabling complications of diabetes
resulted from metabolic defects over a long period.
• 4-hydroxyisoleucine unlike its similarity to isoleucine in the
perspective of molecular structure and hypoglycaemic property,
has wider activities via interacting with pathways which are not
affected by isoleucine. 4-hydroxyisoleucine molecular structure
core is similar to isoleucine, but its extra hydroxyl group at
position four creates unique distinctive properties from
isoleucine.Current in-vitro study results indicate that isoleucine
and 4-hydroxyisoleucine have different modes of action. Its
glucose lowering effect is dependent on glucose concentration
and protein synthesis that suggests a different pathway for 4-
hydroxyisoleucine and could explain the other extra effects of 4-
hydroxyisoleucine in modifying diabetes adverse effects compare
to isoleucine.
150
• 4-hydroxyisoleucine activity is strongly dependent on new protein
synthesis and GLUT1 activity. GLUT1 is widely available in most
of the cells and it controls the basal glucose uptake independent of
insulin. The connection between GLUT1 and 4-hydroxyisoleucine
effect in cellular level, supports the idea that 4-hydroxyisoleucine
utilises the glucose basal consumption and uptake of cells.
• PI3 Kinase plays essential role in the isoleucine mode of action
but not 4-hydroxyisoleucine as proposed in previous literatures.
Increasing of the PI3Kinase activity in tissues treated by 4-
hydroxyisoleucine as observed in previous studies could be an
indirect effect of 4-hydroxyisoleucin.
• Both 4-hydroxyisoleucine and isoleucine activities are strongly
dependent on mitochondrial respiration, but 4-hydroxyisoleucine
up-regulates glycolysis significantly which is not affected by
isoleucine. The connection between mitochondria calcium
signalling and contradictory behaviour of 4-hydroxyisoleucine and
isoleucine in front of reuthenium red support the very important
role of calcium in their mechanisms of action.
• The improving effects of 4hydroxyisoleucine on metabolic
parameters in diabetes including plasma glucose, lipid profile and
uric acid in animal study, and increasing glucose consumption,
glycolysis and mitochondrial activity in BRIN-BD11 cells, suggest
that 4hydroxyisoleucine has a systemic effect on metabolically
active tissues, including the liver, adipose, muscle and pancreatic
beta cells that are independent of insulin. It does not exclude the
role of insulin in combination with 4-hydroxyisoleucine which
151
may amplify its effect as shown previously, but current study
findings exclude the insulin secretion stimulation and interaction
directly with insulin as the key mechanism of action of 4-
hydroxyisoleucine.
• Current study findings indicate that both 4-hydroxyisoleucine and
isoleucine have mechanisms beyond the insulin regulatory
pathway and affect metabolism and energy modulation in both
cytoplasmic and mitochondrial levels which requires more
profound and meticulous study.
We tried to create an overview and basis about potential mechanisms
involved in 4-hydroxyisoleucine in our study to open a window for more
researches in the future. The evaluation of 4-hydroxyisoleucine and
isoleucine on expression of a variety of genes in different types of cell
using microarray technique could provide an immense data about their
mechanisms of action.
4-hydroxyisoleucine is an anti-diabetic molecule with multiple actions,
which is not only reduces plasma glucose in type1 and 2 diabetes, but
modifies other metabolic complications of diabetes and it is only active
in high glucose concentration unlike other antidiabetic medications.
There have been no reports of any adverse effects or toxicity of
4hydroxyisoleucine in previously published studies which indicate that it
is well tolerated and not toxic (Flammang et al. 2004). It makes the 4-
hydroxyisoleucine an intelligent molecule which only active in diabetic
conditions and its consumption do not produce the hypoglycaemic effect
in normoglycaemic or non-diabetic condition. It is a safe choice, unlike
other medications if it is consumed by non-diabetic patients or misused.
Analysing the data from our animal and in-vitro studies determine that 4-
152
hydroxyisoleucine could be a promising candidate for type 1 and 2
diabetes solely or in combination with other therapeutic regimen.
Unfortunately, there is no precise information about the dosage of 4-
hydroxyisoleucine for such application in human. There are some
suggestions based on traditional medicine for using fenugreek seed
extract but there is no verifiable scientific data about the dosage of
fenugreek. The lack of such useful information makes
pharamcokinetic studies an important field to understand 4-
hydroxyisoleucine metabolism in the body, interaction with other
medications and dose response.
5.1 Future recommended studies
There are some ideas for the future studies recommended in this chapter
based on the current study finding to provide a better and clearer
understanding of 4-hydroxyisoleucine mechanisms of action. Carrying
more fundamental and accurate studies which obviously require more
resources and supports to discover 4-hydroxyisoleucine mechanisms of
action which not only allow us to develop more efficient and safer
therapeutics, but also guide us towards a better understanding of the
pathophysiology of diabetes and insulin resistance.
There are a summary of suggested future studies for 4-hydroxyisoleucine
which may be used as ideas for designing better research projects in the
future.
• Investigation of the GLUT 1 expression and its gene transcription
in pancreatic beta cell, liver, adipose and skeletal muscle cell
models incubated with 4-hydroxyisoleucine in the presence of
high glucose concentration. It is suggested to use a dosage titration
to identify the relation of any changes with 4-hydroxyisoleucine.
153
• As we showed the role of calcium in 4-hydroxyisoleucine activity,
it is important to study the role of calcium signalling pathways
within the cell in more details not only in pancreatic beta cell but
in other types of cells incubated with 4-hydroxyisoleucine and
isoleucine for comparison.
• Based on the Seahorse autoanalyzer results which showed
increased activity in both glycolysis and mitochondrial respiration,
It is recommended to look into the interaction of 4-
hydroxyisoleucine with glucokinase and other enzymes within the
Krebs cycle. There is a potential possibility of direct or indirect
interaction between 4-hydroxyisoleucine and Krebs cycle's
enzyme. Investigation of the expression and activity of the Krebs
cycle enzymes in pancreatic beta cell and other cell models such
as liver and adipose could provide a valuable data about the role of
4-hydroxyisoleucine in energy modulation via interacting with
mitochondrial respiration.
• Screening the whole genome expression level in the cell or tissue
models treated with 4-hydroxyisoleucine and isoleucine in front of
high concentration of glucose compared to non treatment control
using a microarray technique could reveal a valuable data about
their mechanisms of action. It also creates a database for different
kind of analysis to provide a wider perceptive about 4-
hydroxyisoleucine and isoleucine activities in gene level.
Unfortunately, carrying such experiment is expensive and requires
substantial resources and facilities.
• A limited human clinical trial could be very informative to
evaluate the efficacy of 4-hydroxyisoleucine in diabetic patients
solely or in combination with other available medication. It
requires pharmacokinetics study prior to clinical study to set out a
154
dose response and more information about 4-hydroxyisoleucine
metabolism. It also requires more cost effective chemistry method
to produce an affordable synthetic 4-hydroxyisoleucine because it
is an expensive and a rare molecule from being used in milligram
dosage for clinical study.
155
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Appendix I
Animal study publication
(Non-insulin dependent anti-diabetic
activity of (2S, 3R, 4S) 4-hydroxyisoleucine
of fenugreek (Trigonella foenum graecum)
in streptozotocin-induced type I diabetic
rats)
192
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Appendix II
4-Hydroxyisoleucine certificate of analysis
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Appendix III
Glucometer accuracy measurements table
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